Esta pesquisa é financiada pelo Henry AACR Bexiga Shepard Cancer Research Grant (09-60-30-LI).

Procedimentos que envolvem seres animais foram aprovados pelo Institutional Animal Care e do Comitê de Uso (IACUC) da Universidade da Califórnia, em San Francisco. 1. Preparação de células <ol><li> A bexiga humana linha de células de cancro KU-7-luc2-GFP (Caliper Life Sciences, Hopkinton, MA) é crescido em meio RPMI-1640 suplementado com 10% de soro fetal de bovino e 50 gentamicina ug / ml. </li><li> As culturas são mantidas a 37 ° C, 5% de CO 2 com os meios de muda a cada dois a três dias. </li><li> As células foram colhidas por digestão com tripsina e contadas utilizando um hemocitómetro. Uma única suspensão celular de 2 x 10 6 células em 50 ul foi preparada em solução salina tamponada com fosfato e mantido em gelo. </li></ol> 2. Modelo de tumor ortotópico de bexiga <ol><li> Fêmeas nu (nu / nu) ratinhos 8-10 semanas de idade (Simonsen Laboratory, Gilory, CA) foram usadas. </li><li> Coloque um pano estéril sobre uma almofada de aquecimento para servir a umsa plataforma cirúrgica. Estéril lubrificante geleia, 24-Guage cateteres com agulhas estilete, solução de nitrato de prata, e água estéril, que são preparados. </li><li> Anestésico geral é induzida nos ratinhos com isoflurano inalado (3% para a indução, 1-2% para manutenção) e mantida através nosecone. Pomada oftálmica estéril é aplicada para os olhos do animal, e uma almofada de calor é utilizado para manter o calor do corpo. </li><li> Posicione o mouse na posição supina. </li><li> Suavemente agarrar vulva do animal com uma pinça, para estabilização. </li><li> Utilizando um cateter 24-guage intravenosa com o estilete agulha removido, a uretra do rato é cateterizada. Lubrificação amplo deve ser usado. Com o supino do mouse, a uretra está localizado imediatamente posterior às pregas vulvares e anterior à vagina. Um ângulo de 45 graus é inicialmente tomadas a fim de canular a uretra e passar por baixo do osso púbico. Um ângulo de mais superficial é então levado a passar através da uretra para a bexiga. Suaves pres pélvicosCertifique-se de endireitar a uretra muitas vezes podem ajudar. Cuidados devem ser tomados para evitar empurrar demasiado duro contra a resistência, pois isso pode levar à perfuração da uretra ou bexiga. Urina visto dentro do cateter confirma a sua localização no interior do lúmen da bexiga. </li><li> Aspirar a urina a partir do lúmen do cateter e da bexiga usando a agulha estilete. A agulha estilete é usado para todas as aspirações subsequentes e injecções, deixando o cateter no lugar. Isto permite a injecção de volumes precisos, eliminando o espaço morto dentro do cateter, e faz cateterismos repetidas desnecessária. </li><li> Para prejudicar a camada de glicosaminoglicano do urotélio da bexiga, 10 ul de 0,5 M de nitrato de prata é injectado e deixou-se habitar durante 10 segundos. Isto pode formar um precipitado branco com urina restante. </li><li> Aspirar e descartar o conteúdo precipitar e bexiga usando uma agulha de estilete. </li><li> Lave a bexiga através da injeção de água 100 uL estéril. Aspirar e descarte a água. Repeem lavagem com água estéril 3 vezes para um total de 4 lavagens. </li><li> Coloque uma gravata de seda 4-0 ao redor do meato uretral para ocluir a uretra na preparação para a remoção do cateter. </li><li> Injectar previamente preparado 2 x 10 6 KU-7-GFP-luc2 células em 50 uL utilizando a agulha de estilete. </li><li> Simultaneamente remover o cateter ea agulha estilete, deixando a uretra ocluída pelo laço de seda. </li><li> O rato será mantida sob anestesia geral, durante 2 horas antes do laço de seda é removido eo animal é acordado. </li></ol> 3. Ultra-som <ol><li> Anestesia geral foi induzida com isoflurano vaporizado e mantida através de cone do nariz. </li><li> A VisualSonics Vevo 770 In Vivo de Alta Resolução Micro-Imaging System (VisualSonics Inc, Toronto, Ontário, Canadá) com a RMV-704 sonda de 40 MHz foi utilizado. </li><li> Posicione o mouse na posição supina na plataforma ultra-som. </li><li> Proteger as patas traseiras do animalcom fita para evitar o movimento excesso durante a manipulação da sonda de ultra-som. </li><li> Aplique o gel de ultra-som à pelve mouse. </li><li> Baixa um RMV-704 sonda de ultra-som (40 MHz) para a bacia do mouse para obter imagens transversais ou longitudinais. </li><li> Se o tumor for encontrado, as imagens podem ser salvas para a medição das dimensões do tumor. </li></ol> 4. Bioluminescent imagem <ol><li> Executar a injecção intraperitoneal de 200 uL (150 mg / kg) substrato luciferina. </li><li> Induzir anestesia geral com isoflurano vaporizado e manter através de cone do nariz. </li><li> Coloque ratos em decúbito dorsal no imager bioluminescência. </li><li> Cinco minutos após a injecção luciferina, medir a bioluminescência a partir do animal em fotões por segundo. </li></ol> 5. Preparação sarna <ol><li> Sintetizar RNA oligonucleotídeos em uma escala de 50-100 mmol. </li><li> Recozer complementares de um único oligonucleótidos de cadeia isaRNAs nto duplex. </li><li> Lipid nanopartícula-(LNP)-sarna são preparados com o lípido ionizável 1,2-dilinoleyl-4-(2 - dimetilaminoetil) - [1,3]-dioxolano (DLinKC2-DMA), disteroylphosphatidyl colina, colesterol, e PEG-DMG usando uma espontânea de vesículas procedimento de formulação de formação, como descrito anteriormente. 1 </li></ol> 6. Administração intravesical de sarna Formulado <ol><li> Depois de estabelecer o modelo de tumor ortotópico bexiga, os ratos são tratados com intravesical pequena activador RNA (sarna) formulada em nanopartículas de lípidos (LNPs) (Tabela 1). </li><li> Anestésico geral é estabelecido e mantido com vaporizado isoflurano, e lubrificante oftálmica estéril é aplicado. </li><li> Coloque o animal em decúbito dorsal. </li><li> Realizar cateterismo uretral como descrito anteriormente. </li><li> Coloque uma gravata de seda 4-0 ao redor da uretra e do cateter. </li><li> Aspirar toda a urina a partir do cateter ebexiga usando uma seringa e agulha vazio estilete. </li><li> Injectar LNP-sarna intravesical para uma dose total de 50 uL (3 mg / kg). Simultaneamente remover o cateter ea agulha estilete, deixando a uretra ocluída pelo laço de seda. </li><li> O rato permanecerá anestesiados com a uretra ocluída durante 2 horas. </li><li> O tratamento com intravesical LNP-sarna foi iniciado no dia 4 após a implantação do tumor. Os ratinhos foram tratados em série, cada 3 dias, para um total de 14 tratamentos. </li></ol> 7. Os resultados representativos Tumores da bexiga decorrentes KU-7-luc2-GFP células pode ser monitorizada por luciferase bioluminescente de imagem (Figura 1A e C) e de ultra-som da bexiga (Figura 1B e D). Os tumores são frequentemente detectável dentro de 3-5 dias após a inoculação das células. Encontramos a implantação do tumor de bexiga com sucesso em mais de 90% dos ratos. Como o tratamento com ARNdc intravesical se segue, a progressão do tumor pode ser monitorizada wiº destas modalidades. Depois de o animal é sacrificado, o crescimento do tumor pode também ser confirmada utilizando GFP de fluorescência. Em geral, a média de bioluminescência correlacionada com dimensões de ultra-som médios para os tumores da bexiga, como visto visualmente nas Figuras 1C e 1D. No entanto, depois de os animais foram sacrificados e tumor foi confirmada por GFP, verificou-se que a bioluminescência correlacionada mais estreitamente com a presença de tumor viáveis ​​do que as medições de ultra-som. Isto foi especialmente aparente entre ratinhos tratados com sarna (em oposição aos controlos), porque o tecido da cicatriz residual em ratinhos tratados era muitas vezes visualizadas por ultra-sons, mas não podia ser diferenciado do tumor ao vivo. <img src="/files/ftp_upload/4207/4207fig1.jpg" alt="A Figura 1" /> Figura 1. Cinética de crescimento de tumor de tumores da bexiga. (A) de imagem bioluminescência mede a actividade da luciferase em tumores da bexiga para confirmar a sua localização e crescimento. (B) In vivo ultrasoun bexigad usando uma sonda de 40MHz produz imagens tumorais, cujas dimensões podem ser medidos com precisão. (C) Os gráficos ilustrando aumento progressivo do tumor em dimensões de bioluminescência e ultra-som. Os valores representam a média (+ / - desvio padrão) medições a partir de 6 animais. <a href="http://www.jove.com/files/ftp_upload/4207/4207fig1large.jpg" target="_blank">Clique aqui para ver maior figura</a> .

<ol>
	<li>Semple, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+cationic+lipids+for+siRNA+delivery.">Rational design of cationic lipids for siRNA delivery.</a> Nature. 28, 172-176 (2010).</li><li>Li, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+dsRNAs+induce+transcriptional+activation+in+human+cells.">Small dsRNAs induce transcriptional activation in human cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 103, 17337-17342 (2006).</li><li>Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antitumor+effect+of+dsRNA-induced+p21(WAF1/CIP1)+gene+activation+in+human+bladder+cancer+cells.">Antitumor effect of dsRNA-induced p21(WAF1/CIP1) gene activation in human bladder cancer cells.</a> Molecular cancer therapeutics. 7, 698-703 (2008).</li><li>Chancellor, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preliminary+results+of+myoblast+injection+into+the+urethra+and+bladder+wall:+a+possible+method+for+the+treatment+of+stress+urinary+incontinence+and+impaired+detrusor+contractility.">Preliminary results of myoblast injection into the urethra and bladder wall: a possible method for the treatment of stress urinary incontinence and impaired detrusor contractility.</a> Neurourology and urodynamics. 19, 279-287 (2000).</li><li>Hadaschik, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+validated+mouse+model+for+orthotopic+bladder+cancer+using+transurethral+tumour+inoculation+and+bioluminescence+imaging.">A validated mouse model for orthotopic bladder cancer using transurethral tumour inoculation and bioluminescence imaging.</a> BJU international. 100, 1377-1384 (2007).</li><li>Fu, C., Apelo, C. A., Torres, B., Thai, K. H., Hsieh, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Bladder+Wall+Injection.">Mouse Bladder Wall Injection.</a> J. Vis. Exp. (53), e2523 (2011).</li><li>Dobek, G. L., Godbey, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Orthotopic+Model+of+Murine+Bladder.">An Orthotopic Model of Murine Bladder.</a> Cancer. J. Vis. Exp. (48), 2535 (2011).</li><li>Dinney, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+metastatic+variants+from+human+transitional+cell+carcinoma+passaged+by+orthotopic+implantation+in+athymic+nude+mice.">Isolation and characterization of metastatic variants from human transitional cell carcinoma passaged by orthotopic implantation in athymic nude mice.</a> The Journal of urology. 154, 1532-1538 (1995).</li></ol>

LCL é um inventor chamado em pedidos de patentes pendentes relacionadas com a sarna que foram arquivados pela Universidade da Califórnia em San Francisco e licenciada para Alnylam Pharmaceuticals.

Apresenta um protocolo para o estabelecimento de um modelo ortotópico xenoenxerto rato tumor da bexiga, seguido de tratamento intravesical dos tumores com LNP sarna formulado que tem como alvo o promotor de p21 CIP1/WAF1 (p21) para a activação transcricional. 2, 3 Os tumores resultantes podem ser confirmada e monitorados com exames de imagem de bioluminescência e ultra-som da bexiga. Modelos ortotópicos pode ser superior à intraperitoneal, subcutânea ou intravenosa os modelos, fornecendo um ...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </td></tr><tr><td> KU-7-luc-GFP células </td><td> Caliper </td><td> 128091 </td><td></td></tr><tr><td> tímicas ratinhos nus (nu / nu) </td><td> Simonsen Laboratories </td><td> 6-7 semanas de idade </td><td> Sim: (NCR) nu / nu fisol </td></tr><tr><td> Unidade de ultra-som </td><td> Visualsonics </td><td> Vevo 770 </td><td> RMV-704 sonda </td></tr><tr><td> Unidade de bioluminescência </td><td> Caliper </td><td> IVIS Spectrum </td><td></td></tr><tr><td> Exel cateter safelet </td><td> Pescador </td><td> 14-841-21 </td><td> 24G x ¾ &quot; </td></tr><tr><td> O nitrato de prata </td><td> Sigma </td><td> S8157 </td><td></td></tr><tr><td> Hamilton seringa </td><td> Hamilton company </td><td> 80301 </td><td></td></tr><tr><td> LNP-sarna </td><td> Alnylam Pharmaceuticals </td><td></td><td></td></tr></tbody></table> Tabela 1. Reagentes específicos e equipamentos.

an orthotopic bladder tumor model and the evaluation of intravesical sarna treatment

Apresentamos um novo método para o tratamento de cancro da bexiga com intravesical entregue pequena activador RNA (sarna) em um xenoenxerto modelo de rato ortotópico tumor da bexiga. O modelo do rato é estabelecido por cateterismo uretral sob anestesia geral inalatória. Queimadura química é então introduzido para a mucosa da bexiga usando solução de nitrato de prata intravesical para romper a camada de glicosaminoglicano bexiga e permite que as células para anexar. Após várias lavagens com água estéril, cancro da bexiga humana KU-7-luc2-GFP células são instilado através do cateter no interior da bexiga para habitar durante 2 horas. Subsequente crescimento de tumores de bexiga é confirmada e monitorado por ultra-som na bexiga vivo e de imagem de bioluminescência. Os tumores são então tratados com intravesical sarna formulados em nanopartículas de lípidos (LNPs). O crescimento do tumor é monitorizado com ultra-sons e bioluminescência. Todos os passos do presente processo são demonstradas no vídeo que o acompanha.

Watch this Scientific Journal Video about An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment at JoVE.com

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

Estabelecer um modelo de tumor de bexiga ortotópico para avaliar os efeitos antitumorais de sarna intravesical entregue e acompanhamento do crescimento tumoral por ultra-som e de imagem de bioluminescência.

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 ...

an-orthotopic-bladder-tumor-model-evaluation-intravesical-sarna

Research

JoVE Journal

Medicine

14.6K visualizações.  University of California, San Francisco. Estabelecer um modelo de tumor de bexiga ortotópico para avaliar os efeitos antitumorais de sarna intravesical entregue e acompanhamento do crescimento tumoral por ultra-som e de imagem de bioluminescência.

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

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 ...

Medicina

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 ...

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 Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

Um modelo de camundongo ortotópico de Carcinoma anaplásico da tiróide

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

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  ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

Minimamente Invasiva Estabelecimento de murino ortotópico bexiga Xenoenxertos

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

Avaliação do tumor infiltrantes Leucócitos Subconjuntos em um modelo de tumor subcutâneo

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold standard treatment for the high-grade non-muscle invasive bladder cancer (NMIBC). However, since the treatment-related side effects are relevant, newer biological response modifiers with a better benefit/side effects ratio are needed.
The tumour microenvironment can influence both tumour development and therapy efficacy. In order to obtain a good model, it is desirable to implant tumour cells in the organ from which the cancer originates.
In this protocol, we describe a method for establishing a tumour in the bladder cavity of female mice and subsequent delivery of therapeutic agents; the latter are exemplified by our use of Helicobacter pylori neutrophil activating protein (HP-NAP). A preliminary chemical burn of the mucosa, followed by the injection of mouse urothelial carcinoma cell line MB49 via urethral catheterization, enables the cells to attach to the bladder mucosa. After a period, required to allow an initial proliferation of the cells, mice are treated with HP-NAP, administrated again via catheterization. The anti-tumour activity of HP-NAP is evaluated comparing the tumour volume, the extent of necrosis and the degree of vascularization between vehicle- and HP-NAP-treated animals.

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.

Avaliação da Eficácia da<em&gt; H. pylori</em&gt; Protein HP-NAP como uma ferramenta terapêutica para o tratamento de cancro da bexiga em um Modelo murino Orthotopic

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

A &quot;Patient-Like&quot; Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

A &quot;Patient-Like&quot; Orthotopic Singênico Rato Modelo de carcinoma hepatocelular Metástase

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...