Esta pesquisa é apoiada pelo financiamento do programa intramuros do NIH.

I. preparação de células <ol><li> As células são crescidas até à confluência em DMEM com FBS 10% sobre um 80% 225 centímetros confluente 3 balão para cada 5 ratinhos a ser implantadas. </li><li> Aspirar meios fora das células; lava-se com 10 ml de PBS sem cálcio ou magnésio. Aspirado PBS células fora. </li><li> Trypsinize células com 3 ml de tripsina, esperar 10-15 min, e neutralizar a tripsina com 15 ml de meio. </li><li> Pipetar todas as células e meios de comunicação em um cónico de 50 ml. Dois frascos grandes caber em um cónico de 50 ml. </li><li> Girar as células a 1600 rpm durante 7 minutos a 4 ° C. Aspirar completamente o sobrenadante. Ressuspender as células em 10 ml de PBS frio, e enxaguar células por suavemente pipetando para cima e para baixo. Assegure-se o pellet celular é misturada uniformemente com o PBS. A suspensão deve ser nublado. </li><li> Neste momento uma contagem de células é feita usando um contador Coulter. As células são novamente centrifugado a 1600 rpm durante 7 minutos a 4 ° C. </li><li> Após a centrifugação nd 2 das células, comopirata todos PBS e ressuspender em 1 ml de PBS. </li><li> As células são centrifugadas a em 1 ml de PBS a 3000 rpm durante 10 min a 4 ° C. O PBS é aspirado, e PBS é adicionado para ajustar a concentração final de 1x10 6 por 5 uL para U87 células, outras linhas celulares podem exigir um número diferente e concentração de células para o crescimento do tumor óptima. </li><li> Após o volume final e concentração obtém-se as células são mantidas em gelo em um tubo de Eppendorf. </li></ol> II. Xenoenxerto <ol class="ualpha"><li> Preparando o mouse <ol><li> Os ratos são anestesiados com uma mistura de injecção de Cetamina / Xilazina / solução salina. A mistura é de 120 mg / kg de cetamina, 16 mg / kg de Rompun e solução salina. Os ratinhos são pesados ​​individualmente e deu 0,1 ml por 10 g com base no seu peso. O anestésico é dado com agulha 26G ligado a uma seringa IP ml. Levará cerca de 15 min para os ratos para chegar a uma fase de sono sem resposta à dor. Você pode verificar a resposta à dor dacamundongos por beliscar a sua pata. </li><li> Após o mouse está totalmente anestesiado, pomada é administrado aos seus olhos, para evitar que sequem. </li><li> O rato é colocado sobre o leito do estereotáxico com os dentes da frente superiores no entalhe na barra de boca. O leito é então colocado no estereotáxica. </li><li> A guarda nariz é colocada sobre o nariz do rato para impedir o movimento da cabeça, e as barras de orelha são suavemente colocar nos canais de orelha dos ratinhos. Tenha cuidado para não fazer qualquer colocação de barra de nariz ou barras de ouvido muito apertados, como o crânio podem fraturar e / ou impedir o mouse de respirar. </li><li> Certifique-se que a cabeça do rato é o nível usando as marcas de escala na estereotáxica é útil durante este processo. </li><li> Para preparar o álcool de iodo rato, e providine é aplicada liberalmente sobre a cabeça do rato alternada para um total de 3 aplicação de álcool e 3 aplicações de iodo. Uma aplicação final de uma almofada de álcool com 70% é aplicado paraa cabeça. </li></ol></li><li> Procedimento cirúrgico <ol><li> Todos os instrumentos o procedimento deve ser esterilizado quer por autoclavagem ou embebendo instrumentos em etanol a 70% antes de iniciar o procedimento. </li><li> É feita uma incisão no topo do crânio por trás do olho direito diagonalmente de volta para o posterior esquerda do crânio. A incisão deve ser de cerca de 10 mm a 15 mm de comprimento. </li><li> Usando um Q-Tip, separar lentamente a incisão para expor o crânio e enxugar a membrana que está entre o crânio ea pele. Use um Q-ponta para apagar qualquer vestígio de sangue e assistir a sangramento excessivo. </li><li> Expor o topo do crânio para visualizar a bregma. Utilizando o ponto de seringa Hamilton, posicionar sobre o bregma. A posição correcta do orifício a ser perfurado no crânio é de 2 mm direita e 1 mm anterior da bregma (figura 2). Um marcador cirurgião ponta de feltro, que é comprado estéril, é utilizado para marcar a posição do orifício a ser perfurado. Por favor, note que o buraco está localizado próximo à umalinha de sutura terior. </li><li> Em seguida, faça um pequeno furo no crânio, na posição marcada com a ponta de uma agulha 22G em um vaso de pino ou alimentado através de uma agulha de 18G. </li><li> Carregar o 10 uL de vidro Hamilton seringa com 5 ul de células. A seringa de Hamilton é 10 ul com um contundente 30G para 33G agulha fixa ligada a ela. Certifique-se de misturar as células antes de carregá-las, seja por pipetagem-los ou usando uma seringa de 1 ml com agulha de 25G conectado. Depois de a seringa Hamilton é carregado, colocá-lo de volta no braço para alinhá-lo com o furo no crânio. </li><li> Abaixar a fim bunt da agulha para a superfície do crânio, uma vez que a agulha esteja nivelada com o crânio; baixar a agulha até que seja a uma profundidade de 3 mm abaixo da superfície do crânio. </li><li> Depois de a agulha é reduzida, deve permanecer ali durante 2 minutos para evitar qualquer traumatismo grave da agulha que afecta o cérebro. Implantação das células é a uma taxa de 1 uL por minuto para um total de 6-8 minutos, ter o cuidado de qualquer backflow. Após a última das células terem sido implantadas, deixar a agulha no lugar por mais 2 minutos, para permitir todas as células para resolver dentro </li><li> Retire a agulha do cérebro, e limpe o buraco com um Q-tip. Continuar a limpar a agulha e seringa com 50% acetona, EtOH a 100%, e de PBS, 3x em cada solução e, nessa ordem. </li><li> A pele sobre o crânio está pronto para ser suturado neste ponto. Usando 5-0 PBS fechar a incisão com cerca de 3 pontos. </li><li> Permitir o mouse para acordar em uma almofada de calor, e reaplicar pomada, se necessário. O rato pode ser devolvido à gaiola após mostra movimento na sua própria (isto é, Levantando a cabeça ou movendo os ombros). </li></ol></li></ol> III. O tratamento do implante IC por radiação e de drogas <ol class="ualpha"><li> O tratamento de radiação <ol><li> Dependendo da linha de células, o tratamento dos ratos pode ter lugar de 5 a 7 dias após o implante. Nosso departamento utiliza uma unidade de radioterapia para tratamento de ortovoltagem radiaçãomento. </li><li> Imagiologia, tais como BLI (Imaging bioluminescência), MRI ou CT pode ser usado para visualizar o tumor antes da randomização (figura 3A). </li><li> Os ratos são anestesiados com o cocktail de Cetamina / Xilazina / solução salina como antes, e pomada oftálmica é aplicado aos seus olhos. </li><li> Os ratinhos são posicionados em um jig radiação feitos que está configurado como uma roda de pizza, em que as cabeças dos ratinhos apontar em direcção ao centro. Os corpos dos ratos são protegidos da radiação com chumbo. </li><li> O tratamento medicamentoso pode ser dado antes ou radiação através de rotas de entrega diferentes, SQ ou seja, IP e IV. </li></ol></li></ol> IV. Resultados O rato deve acordar o procedimento dentro de 45-60 minutos após a injeção de anestesia. Se o mouse tem dificuldade de acordar após o procedimento e tempo ultrapassou 1 ½ horas após o procedimento, o mouse pode não recuperar e eutanásia pode ser uma alternativa eficaz. Se there é sangramento excessivo ou o crânio rachado torna-se a qualquer momento durante o procedimento de eutanásia é aconselhada. Se tudo correr bem e, dependendo da linha celular, um tumor deve começar a formar dentro de 2-4 semanas após o procedimento tem lugar. Ao usar modelos xenograft intracranianos, a eficácia terapêutica é geralmente medido como a sobrevivência pós-implantação-5. No entanto, como este é um modelo IC sinais e sintomas ocorrerá antes da morte. Alguns dos sinais físicos do mouse podem apresentar são uma postura arqueada, a perda de peso e inatividade. Alguns dos sinais neurológicos podem ser convulsões, incline a cabeça e perda de equilíbrio. A Figura 3B mostra uma mancha de HE um tumor em mortalidade. Um diagrama de Kaplan-Meier é usado para avaliar os dados (Figura 4). O número de dias de sobrevivência de 50% são calculados e comparados com o controle. Se o grupo de combinação viveram mais do que a adição da radiação indivíduo e os braços de drogas a combinação é foi bem sucedida. <img alt = &quot;Figura 1&quot; src = &quot;files/ftp_upload/3397/3397fig1.jpg /&quot; /&gt; Figura 1. O equipamento Stoelting estereotáxica. <img alt="A Figura 2" src="/files/ftp_upload/3397/3397fig2.jpg" /> Figura 2. Um diagrama da localização do furo de perfurador no crânio do rato. <img alt="A Figura 3A" src="/files/ftp_upload/3397/3397fig3A.jpg" /> Figura 3A. BLI imagem após a implantação do GBM. <img alt="A Figura 3B" src="/files/ftp_upload/3397/3397fig3B.jpg" /> Figura 3B. H e E do tumor GBM a mortalidade. <img alt="A Figura 4" src="/files/ftp_upload/3397/3397fig4.jpg" /> Figura 4. Resultados de U87 implantes IC injetados com inibidor de PARP 7 pós-cirurgia dias.

<ol>
	<li>Bleau, A. M., Holland, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trapping+the+mouse+genome+to+hunt+human+alternations.">Trapping the mouse genome to hunt human alternations.</a> Proc. Nat. Acad. Sci. U.S.A. 104, 7737-7738 (2007).</li><li>McGrady, B. J., McCormick, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+murine+model+of+intracranial+invasion:+morphological+observations+on+central+nervous+system+invasion+by+melanoma+cells.">A murine model of intracranial invasion: morphological observations on central nervous system invasion by melanoma cells.</a> Clin. Exp. Metastasis. 10, 387-393 (1992).</li><li>Fei, X., Zhang, Q., Dong, J., Diao, y., Wang, Z., Li, R., Wu, Z., Wang, A., Lan, Q., Zhang, S., Huang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+clinically+relevant+orthotopic+xenograft+mouse+model+of+metastatic+lung+cancer+and+glioblastoma+through+surgical+tumor+tissues+injection+with+trocar.">Development of clinically relevant orthotopic xenograft mouse model of metastatic lung cancer and glioblastoma through surgical tumor tissues injection with trocar.</a> Journal of Experimental &#38; Clinical Cancer Research. 29, 84-84 (2010).</li><li>Fujita, M., Zhu, X., Sasaki, K., Ueda, R., Low, K., Pollack, I., Okada, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+STAT3+Promotes+the+Efficacy+of+Adoptive+Transfer+Therapy+Using+Type-1+CTLs+by+Modulation+of+the+Immunological+Microenvironment+in+a+Murine+Intracranial+Glioma.">Inhibition of STAT3 Promotes the Efficacy of Adoptive Transfer Therapy Using Type-1 CTLs by Modulation of the Immunological Microenvironment in a Murine Intracranial Glioma.</a> J. Immunol. 180, 2089-2098 (2008).</li><li>Candolfi, M., Curtin, J. F., Nichols, W. S., Muhammad, A. K. M. .. . G., King, G., Pluhar, G. D., McNiel, G. E., Ohlfest, E. A., Freese, J. R., Moore, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+glioblastoma+models+in+preclinical+neuro-oncology:+neuropathlogical+characterization+and+tumor+progression.">Intracranial glioblastoma models in preclinical neuro-oncology: neuropathlogical characterization and tumor progression.</a> J. Neurooncol. 85, 133-148 (2007).</li><li>Kaye, A., Morstyn, G., Gardner, I., Pyke, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+xenograft+glioma+model+in+mouse+brain.">Development of a xenograft glioma model in mouse brain.</a> Cancer Research. 46, 1367-1373 (1986).</li><li>Zhao, Y., Xiao, A., diPierro, C., Carpenter, J., Abdel-Fattah, R., Redpath, G., Lopez, M., Hussaini, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Extensive+Invasive+Intracranial+Human+Glioblastoma+Xenograft+Model.">An Extensive Invasive Intracranial Human Glioblastoma Xenograft Model.</a> American Journal of Pathology. 176, 3032-3049 (2010).</li><li>Learn, C., Grossi, P., Schmittling, R., Xie, W., Mitchell, D., Karikari, I., Wei, Z., Dressman, H., Sampson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Analysis+of+Intracranial+Tumors+in+a+Murine+Model+of+Glioma+Demonstrate+a+Shift+in+Gene+Expression+in+Response+to+Host+Immunity.">Genetic Analysis of Intracranial Tumors in a Murine Model of Glioma Demonstrate a Shift in Gene Expression in Response to Host Immunity.</a> J. Neuroimmunol. 182, 63-72 (2007).</li><li>Martens, T., Laabs, Y., Günther, H., Kemming, D., Zhu, Z., Witte, L., Hagel, C., Westphal, M., Lamszus, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Glioblastoma+Growth+in+a+Highly+Invasive+Nude+Mouse+Model+Can+Be+Achieved+by+Targeting+Epidermal+Growth+Factor+Receptor+but+not+Vascular+Endothelial+Growth+Factor+Receptor-2.">Inhibition of Glioblastoma Growth in a Highly Invasive Nude Mouse Model Can Be Achieved by Targeting Epidermal Growth Factor Receptor but not Vascular Endothelial Growth Factor Receptor-2.</a> Clin. Cancer Res. 14, 5447-5458 (2008).</li><li>Purow, B., Schiff, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+genetics+of+glioblastoma:+are+we+reaching+critical+mass.">Advances in the genetics of glioblastoma: are we reaching critical mass.</a> Nat. Rev. Neurol. 5, 419-426 (2009).</li></ol>

Não temos nada a divulgar.

Gliomas malignos, como o glioblastoma multiforme, representam os mais comuns tumores cerebrais primários e têm um prognóstico sombrio 4. Sobrevida de pacientes afetados por GBM permaneceu praticamente inalterada durante a última década (isto é, 9-12 meses após o diagnóstico), apesar dos avanços em cirurgia, radioterapia e quimioterapia 5. As características de um modelo adequado para o estudo do tratamento glioma deve incluir uma localização do tumor reprodutível e crescimento das cél...

<table border="1"><tbody><tr><td> Laboratório Padrão Estereotáxica Instrutment </td><td> Stoelting Mouse e adaptador rato </td></tr><tr><td> Betadine </td><td> 100% de EtOH </td></tr><tr><td> 70% de EtOH </td><td> Acetona 50% </td></tr><tr><td> 6 &quot;Q-tips </td><td> Compressas com álcool </td></tr><tr><td> Hamilton seringa 80008 </td><td> Ethicon 5-0 PD </td></tr><tr><td> 1701 SN 31-33G / 1 &quot;/ PT3 agulha </td><td> Seringas de 1 ml </td></tr><tr><td> Agulhas 25G </td><td> Almofada de aquecimento </td></tr><tr><td> Pomada Lágrimas Artificial </td><td> Muito bom marcador permanente ponta </td></tr></tbody></table>

combination radiotherapy in an orthotopic mouse brain tumor model

O glioblastoma multiforme (GBM) são os mais comuns e agressivos adultos tumores cerebrais primários 1. Em anos recentes, tem havido um progresso substancial na compreensão dos mecanismos de invasão do tumor, e inoculação intracerebral directa de tumor proporciona a oportunidade de se observar o processo invasivo em um ambiente apropriado fisiologicamente 2. No que diz respeito tumores cerebrais humanos estão em causa, os modelos ortotópicos actualmente disponíveis são estabelecidas, quer por injecção estereotáxica de suspensões de células ou implantação de um pedaço sólido de tumor através de um procedimento complicado craniotomia 3. Na nossa técnica que colher células de cultura de tecidos para criar uma suspensão de células usadas para implante directamente para o cérebro. A duração da cirurgia é aproximadamente 30 minutos, e como o rato precisa de ser constante num plano cirúrgico, um anestésico injectável é usado. O rato é colocado em um jig estereotáxico feita por Stoetling (figura 1). À réer a área cirúrgica é limpo e preparado, é feita uma incisão; eo bregma está localizado para determinar a localização do craniotomia. A localização do craniotomia é de 2 mm para a direita e 1 mm rostral à bregma. A profundidade é de 3 mm da superfície do crânio, e as células são injectadas a uma taxa de 2 ul a cada 2 minutos. A pele é suturada com 5-0 PDS, e do rato é permitido para acordar em uma almofada de aquecimento. De nossa experiência, dependendo da linha celular, o tratamento pode ter lugar a partir de 7-10 dias após a cirurgia. A entrega da droga depende da composição de droga. Para o tratamento de radiação os ratos são anestesiados e colocados em um feito gabarito. Chumbo cobre o corpo do rato e expõe apenas o cérebro do rato. O estudo de tumorigénese ea avaliação de novas terapias para GBM requerem precisos e reprodutíveis cerebrais modelos animais de tumores. Assim, utilizar este modelo ortotópico cérebro para estudar a interacção do microambiente do cérebro e do tumor, para testar a effectiveness de diferentes agentes terapêuticos, com e sem radiação.

Watch this Scientific Journal Video about Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model at JoVE.com

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

O objetivo deste artigo é descrever o uso de um modelo de glioblastoma ortotópico para estudos quimioradioterapia. Este artigo irá passar por transformação celular, implantação, e radioterapia do mouse através de um modelo intracraniana.

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 ...

combination-radiotherapy-in-an-orthotopic-mouse-brain-tumor-model

Research

JoVE Journal

Medicine

16.3K visualizações.  National Cancer Institute. O objetivo deste artigo é descrever o uso de um modelo de glioblastoma ortotópico para estudos quimioradioterapia. Este artigo irá passar por transformação celular, implantação, e radioterapia do mouse através de um modelo intracraniana.

Vídeo: Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

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 ...

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

Na imagem de bioluminescência vivo Tumor de mama Dynamics Hipóxia metástase do cancro cerebral em um modelo de rato

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

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 ...

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 ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

A Novel de alta resolução<em&gt; In vivo</em&gt; Técnica de Imagem para estudar a resposta dinâmica de estruturas intracranianas ao crescimento do tumor e Terapêutica

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

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  ...

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

Um Glioblastoma Rato modelo ortotópico Manter restrições Cérebro parênquima físicas e Adequado para intravital dois fótons Microscopia

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

Tumor Treating Field Therapy in Combination with Bevacizumab for the Treatment of Recurrent Glioblastoma

A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA approved in April 2011 for the treatment of patients 22 years or older with rGBM. The device delivers alternating electric fields and is programmed to ensure maximal tumor cell kill1.
Glioblastoma is the most common type of glioma and has an estimated incidence of approximately 10,000 new cases per year in the United States alone2. This tumor is particularly resistant to treatment and is uniformly fatal especially in the recurrent setting3-5. Prior to the approval of the TTF System, the only FDA approved treatment for rGBM was bevacizumab6. Bevacizumab is a humanized monoclonal antibody targeted against the vascular endothelial growth factor (VEGF) protein that drives tumor angiogenesis7. By blocking the VEGF pathway, bevacizumab can result in a significant radiographic response (pseudoresponse), improve progression free survival and reduce corticosteroid requirements in rGBM patients8,9. Bevacizumab however failed to prolong overall survival in a recent phase III trial26. A pivotal phase III trial (EF-11) demonstrated comparable overall survival between physicians&#8217; choice chemotherapy and TTF Therapy but better quality of life were observed in the TTF arm10.
There is currently an unmet need to develop novel approaches designed to prolong overall survival and/or improve quality of life in this unfortunate patient population. One appealing approach would be to combine the two currently approved treatment modalities namely bevacizumab and TTF Therapy. These two treatments are currently approved as monotherapy11,12, but their combination has never been evaluated in a clinical trial. We have developed an approach for combining those two treatment modalities and treated 2 rGBM patients. Here we describe a detailed methodology outlining this novel treatment protocol and present representative data from one of the treated patients.

A novel methodology that is employed for the treatment of recurrent glioblastomas is described. This treatment approach employs the application of alternating electric tumor treating fields (TTFields), known as TTF Therapy in combination with bevacizumab, a targeted agent that is currently FDA approved as monotherapy.

Tumor Tratar Terapia campo em combinação com bevacizumabe para o tratamento do glioblastoma recorrente

A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA ...