Name: utilization of ultrasound guided tissue-directed cellular implantation for the establishment of biologically relevant metastatic tumor xenografts
Uploaded: 2018-05-25T14:30:00
Description: Watch this Scientific Journal Video about Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts at JoVE.com

Este trabalho recebeu apoio a Robert Wood Johnson Foundation/Amos faculdade desenvolvimento programa, Taubman Instituto de investigação médica e a seção de cirurgia pediátrica, a Universidade de Michigan. Os autores desejam agradecer a Kimber iniciantes-Borges e Dr. Marcus Jarboe para a assistência com procedimentos de injeção de ultra-som e a plataforma de imagem. Agradecemos a Paul Trombley por sua ajuda com os gráficos da figura. Agradecemos também o departamento de Radiologia da Universidade de Michigan para a utilização do centro de imagem Molecular e o núcleo do Tumor Imaging, sustentados em parte por abrangente Cancer Center NIH, conceda P30 CA046592. A Universidade de Michigan fisiologia fenotipagem Core que é suportado em parte pela concessão de financiamento do NIH (OD016502) e do centro do sistema circulatório de Frankel. Autenticação de linha celular foi feita em instalações de bio-investigação IDEXX RADIL, Columbia, MO. Agradecemos a Tammy Stoll, Dr. Rajen Mody e o programa de Oncologia Tumor sólido Mott. Nossos pacientes e familiares com gratidão são reconhecidos por sua inspiração, coragem e apoio permanente da nossa pesquisa.

Todo o trabalho foi feito de acordo com o Conselho de revisão institucional da Universidade de Michigan (HUM 00052430) e está de acordo com procedimentos aprovados pelo Comitê de Universidade no uso e cuidado dos animais (UCUCA). A unidade para o laboratório de medicina Animal (ULAM) supervisionou cuidados com animais.
Todo o trabalho foi feito com a aprovação do Conselho de revisão institucional da Universidade de Michigan (HUM 00052430) e está em conformidade com todos os regulamento...

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biologically+relevant+orthotopic+neuroblastoma+xenograft+models:+Primary+adrenal+tumor+growth+and+spontaneous+distant+metastasis.">Biologically relevant orthotopic neuroblastoma xenograft models: Primary adrenal tumor growth and spontaneous distant metastasis.</a> In Vivo. 16 (2), 77-85 (2002).</li><li>Stewart, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+a+human+orthotopic+neuroblastoma+xenograft.">Development and characterization of a human orthotopic neuroblastoma xenograft.</a> Dev Biol. 407, 344-355 (2015).</li><li>J&#228;ger, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimally+Invasive+Establishment+of+Murine+Orthotopic+Bladder+Xenografts.">Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts.</a> J. 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Guiada por ultra-som de implantação de células NB e ES é um método eficiente e seguro para estabelecer confiança xenografts murino para estudos pré-clínicos em biologia do câncer. Críticos para o sucesso de guiada por ultra-som implantação de tecido-alvo é a presença e a disponibilidade de pessoal treinado com conhecimentos especializados em localizar anatomicamente o órgão de interesse e na injeção estereotáxica de células tumorais.
A dissociação do tecido do tumor provo...

Modelos animais xenoenxertos são ferramentas essenciais para estudos pré-clínicos de romance terapias anticâncer. Xenografts murino padrão dependem de implantação subcutânea de flanco das células, fornecendo um local facilmente acessível e eficiente para monitorar o crescimento do tumor. A desvantagem dos modelos subcutâneos é a falta de características biológicas específicas do tumor, que pode limitar seu potencial de metástase1. Tais limitações são superadas pelo uso de xenografts ortotópico no qual tumor células estão incorporadas nos sítios de tecidos nativos, proporcionando um microambiente relevante com potencial metastático2. Modelos de transplante ortotópico mantêm características biológicas originais e fornecem modelos de confiança para descoberta de drogas pré-clínicos3,4. As células cancerosas utilizadas para implantação de tecido-dirigido são linhagens celulares estabelecidas ou paciente-derivado de células de tumores de pacientes. Xenografts estabelecidos de linhas de células de câncer podem apresentar alta divergência genética do tumor primário em relação ao paciente xenografts derivada5. Diante disso, o estabelecimento de paciente-derivado ortotópico xenografts tornou-se o padrão preferencial para teste novela terapêutica na descoberta da droga de cancro.
No neuroblastoma de câncer pediátrico (NB), modelos de transplante ortotópico recapitular a biologia do tumor primário em desenvolvem metástases para locais típicos de NB espalhar6,7. NB se desenvolve na glândula adrenal ou ao longo da cadeia simpática paravertebral. Os métodos mais comuns de implantação ortotópico requerem procedimentos cirúrgicos trans-abdominal abertos. Tais métodos são muitas vezes tediosos, têm alta taxa de mortalidade animal e períodos de recuperação complexa. Ultra-som de alta resolução tem sido recentemente utilizada para tecido-dirigido a implantação de células tumorais no desenvolvimento de vários modelos de murino para câncer pesquisa8,9. A técnica é confiável, pode ser reproduzido, eficiente e segura para o estabelecimento de tumor metastático relevantes xenografts10,11.
O estabelecimento de xenografts câncer pediátrico por alvo guiada por ultra-som órgão localização e agulha implantação de linhas de células e células tumorais do paciente-derivado é demonstrada11. A técnica foi utilizada para NB direcionado para a glândula adrenal murino. Sarcoma de Ewing (ES) é predominantemente um câncer ósseo, comumente visto em ossos longos como fêmur e ossos pélvicos12. Relatos de casos têm demonstrado que, para determinar se o crescimento de um câncer ósseo predominantemente é viável no tecido renal, uma localização capsular sub renal foi escolhida para ortotópico implantação13. Implantação de célula capsular sub renal de células do tumor tem sido utilizada como um modelo promissor para estudar metástases espontâneas para ES14.

<table border="0" cellpadding="0" cellspacing="0" width="1053">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Mice</td>
			<td style="width:231px;"></td>
			<td style="width:232px;"></td>
			<td style="width:295px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NOD-SCID</td>
			<td>Charles River</td>
			<td>394</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NSG</td>
			<td>The Jackson Laboratory</td>
			<td>5557</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Cell Line&#160;</td>
			<td style="width:231px;"></td>
			<td style="width:232px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">NB</td>
			<td style="width:231px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">IMR-32</td>
			<td style="width:231px;">ATCC</td>
			<td>CCL-127</td>
			<td>Established human neuroblastoma cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">SH-SY5Y</td>
			<td style="width:231px;">ATCC</td>
			<td style="width:232px;">CRL-2266</td>
			<td>Established human neuroblastoma cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">SK-N-Be2</td>
			<td style="width:231px;">ATCC</td>
			<td style="width:232px;">CRL-2271</td>
			<td>Established human neuroblastoma cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">ES</td>
			<td style="width:231px;"></td>
			<td style="width:232px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TC32&#160;</td>
			<td style="width:231px;">COGcell.ORG</td>
			<td style="width:232px;"></td>
			<td>Established human Ewing&#39;s Sarcoma cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A673</td>
			<td style="width:231px;">COGcell.ORG</td>
			<td style="width:232px;"></td>
			<td>Established human Ewing&#39;s Sarcoma cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CHLA-25</td>
			<td style="width:231px;">COGcell.ORG</td>
			<td style="width:232px;"></td>
			<td>Established human Ewing&#39;s Sarcoma cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">A4573</td>
			<td style="width:231px;">COGcell.ORG</td>
			<td style="width:232px;"></td>
			<td>Established human Ewing&#39;s Sarcoma cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Cell Line media</td>
			<td style="width:231px;"></td>
			<td style="width:232px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">RPMI</td>
			<td style="width:231px;">Life Technologies</td>
			<td style="width:232px;">11875-093</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Matrigel</td>
			<td style="width:231px;">BD BioSciences</td>
			<td>354234</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Dissociation</td>
			<td style="width:231px;"></td>
			<td style="width:232px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Dissection Tools</td>
			<td style="width:231px;">KentScientific</td>
			<td style="width:232px;">INSMOUSEKIT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Human Tumor Dissociation Kit&#160;</td>
			<td style="width:231px;">MACS Miltenyi Biotec</td>
			<td style="width:232px;">130-095-929</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">gentleMACS dissociator</td>
			<td style="width:231px;">MACS Miltenyi Biotec</td>
			<td>130-093-235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">gentleMACS C tubes</td>
			<td style="width:231px;">MACS Miltenyi Biotec</td>
			<td>130-096-334</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Cell Strainer</td>
			<td style="width:231px;">Corning</td>
			<td>431751</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Luciferase Tagging</td>
			<td style="width:231px;"></td>
			<td style="width:232px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Lenti-GFP1 virus</td>
			<td style="width:231px;">University of Michigan, Vector Core</td>
			<td style="width:232px;"></td>
			<td>Luciferase Virus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Steady Glo-Luciferase Assay Kit</td>
			<td style="width:231px;">Promega</td>
			<td>E2510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Bioluminescence Imaging</td>
			<td style="width:231px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Ivis Spectrum Imaging System</td>
			<td style="width:231px;">PerkinElmer</td>
			<td>124262</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">D-Luciferin</td>
			<td style="width:231px;">Promega</td>
			<td>E160X</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Anesthetic</td>
			<td style="width:231px;"></td>
			<td style="width:232px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Inhaled Isoflurane&#160;</td>
			<td style="width:231px;">Piramal Critical Care Inc</td>
			<td style="width:232px;">66794-0017-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Ultrasound Guided Injection</td>
			<td style="width:231px;"></td>
			<td style="width:232px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Vevo 2100 High Resolution Imaging</td>
			<td style="width:231px;">Vevo</td>
			<td style="width:232px;">2100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Hamilton Syringes (27 gauge needle)</td>
			<td style="width:231px;">Hamilton</td>
			<td style="width:232px;">80000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">22 Gauge Angiocatheter</td>
			<td style="width:231px;">BD Biosciences</td>
			<td style="width:232px;">381423</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Optical ointment</td>
			<td style="width:231px;">Major Pharmaceuticals</td>
			<td style="width:232px;">301909</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Nair</td>
			<td style="width:231px;">Church &#38; Dwight Co</td>
			<td style="width:232px;"></td>
			<td style="width:295px;">Hair Removal agent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Aquasonic 100 Ultrasound Transmission gel</td>
			<td style="width:231px;">Parker</td>
			<td style="width:232px;"></td>
			<td>Ultrasound gel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Histology</td>
			<td style="width:231px;"></td>
			<td style="width:232px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">CD99</td>
			<td style="width:231px;">DAKO</td>
			<td>M3601</td>
			<td>Primary Antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Tyrosine Hydroxylase</td>
			<td style="width:231px;">Sigma-Aldrich</td>
			<td>T2928</td>
			<td>Primary Antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Secondary HRP-Polymer antibody</td>
			<td style="width:231px;">Biocare</td>
			<td>BRR4056KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Miscelleneous</td>
			<td style="width:231px;"></td>
			<td style="width:232px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">10 mL Pipettes</td>
			<td style="width:231px;">Fisher Scientific</td>
			<td style="width:232px;">13-676-10J</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">5 mL Pipettes</td>
			<td style="width:231px;">Fisher Scientific</td>
			<td style="width:232px;">13-676-10H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">1.5 mL Microcentrifuge tubes</td>
			<td style="width:231px;">Fisher Scientific</td>
			<td style="width:232px;">05-408-129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">P1000 pipette</td>
			<td style="width:231px;">Eppendorf</td>
			<td style="width:232px;">3120000062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">P200 pipette</td>
			<td style="width:231px;">Eppendorf</td>
			<td style="width:232px;">3120000054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">P1000 pipette tips</td>
			<td style="width:231px;">Fisher Scientific</td>
			<td style="width:232px;">21-375E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">P200 pipette tips</td>
			<td style="width:231px;">Fisher Scientific</td>
			<td style="width:232px;">21-375D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Portable pipette aid</td>
			<td style="width:231px;">Drummond</td>
			<td>4-000-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">digital animal Weighing Scale&#160;</td>
			<td style="width:231px;">KentScientific</td>
			<td style="width:232px;">SCL-1015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Calipers</td>
			<td style="width:231px;">Fisher Scientific</td>
			<td style="width:232px;">06-664-16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">6well low attachment plates</td>
			<td style="width:231px;">Corning</td>
			<td style="width:232px;">07-200-601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">10 cm Tissue Culture Treated Dishes</td>
			<td style="width:231px;">Fisher Scientific</td>
			<td style="width:232px;">FB012924</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Polybrene</td>
			<td style="width:231px;">Sigma-Aldrich</td>
			<td style="width:232px;">TR-1003-G</td>
			<td></td>
		</tr>
	</tbody>
</table>

utilization of ultrasound guided tissue-directed cellular implantation for the establishment of biologically relevant metastatic tumor xenografts

Testes pré-clínicos de terapias anticâncer baseia-se em modelos de xenoenxertos relevantes que imitam as tendências inatas de câncer. Vantagens dos modelos padrão flanco subcutânea incluem facilidade processual e a capacidade de resposta sem imagens invasivas e progressão do tumor do monitor. Tais modelos são muitas vezes inconsistentes em ensaios clínicos translacionais e limitaram características biologicamente relevantes com baixa propensão para produzir metástase, como há uma falta de um microambiente nativo. Em comparação, modelos de transplante ortotópico em localizações tumorais nativos foram mostrados para imitar o microambiente do tumor e replicar características importantes doenças tais como a propagação metastática distante. Estes modelos geralmente exigem procedimentos cirúrgicos tediosos com períodos prolongados de tempo e recuperação anestésicos. Para resolver isso, pesquisadores de câncer recentemente tem utilizado técnicas de injeção guiada por ultra-som para estabelecer modelos de enxerto heterólogo câncer para experimentos pré-clínicos, que permite a criação rápida e fiável de modelos murino tecido-dirigido. Visualização de ultra-som também fornece um método não invasivo para avaliação longitudinal da enxertia de tumor e crescimento. Aqui, descrevemos o método de injeção guiada por ultra-som de células de câncer, utilizando a glândula adrenal para NB e cápsula renal sub para ES. Esta abordagem minimamente invasiva supera a implantação de cirurgia aberta tedioso de células cancerosas em locais específicos do tecido para o crescimento e a metástase e períodos de recuperação mórbida de abates. Descrevemos a utilização de linhagens celulares estabelecidas e de linhas de células derivadas paciente ortotópico injectável. Pre-feitos jogos comerciais estão disponíveis para dissociação de tumor e de luciferase marcação de células. Injeção da suspensão de células usando imagem-orientação fornece uma plataforma minimamente invasiva e pode ser reproduzida para a criação de modelos pré-clínicos. Este método é utilizado para criar modelos pré-clínicos confiáveis para outros tipos de câncer como bexiga, fígado e pâncreas, exemplificando seu potencial inexplorado de numerosos modelos de câncer.

Usando os procedimentos apresentados, guiada por ultra-som implantação de células NB na glândula adrenal foi feita em uma sala de procedimento dedicado equipada com uma mesa cirúrgica aquecida. Almofadas de braço e no pé foram colocadas para monitorar a atividade do coração murino (figura 1A). O animal permaneceu anestesiado com isoflurano usando inalação do cone de nariz. O rim esquerdo usando uma sonda de ultra-som de alta resolução, foi identi...

Watch this Scientific Journal Video about Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts at JoVE.com

Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts

Aqui, apresentamos um protocolo utilizar injeção guiada por ultra-som de neuroblastoma (NB) e células (ES) de sarcoma de Ewing (estabeleceu linhas celulares e células tumorais do paciente-derivado) biologicamente relevantes locais para criar modelos pré-clínicos confiáveis para câncer pesquisa.

Utilização do ultra-som guiado implantação celular tecido-dirigido para o estabelecimento de Xenografts biologicamente relevantes Tumor metastático

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard ...

The goal of this method is to establish biologically relevant cancer orthotopic xenografts for preclinical studies. Patient-derived neuroblastoma cells are injected into the murine adrenal gland by ultrasound guidance without open surgery or prolonged recovery. This method can help answer critical questions in cancer biology and in translational research, such as studies of tumor evolution, therapeutic responses in the native microenvironment and metastases.Such a knowledge would greatly improve the reliability of preclinical studies and facilitate drug discovery. The main advantage of this technique is that it is patient-derived, tissue-directed, efficient, and reliable in producing an orthotopic xenographed model for cancer therapy research. Demonstrating the critical steps in the procedure will be Sahiti Chukkapalli, a senior technician and our lab manager, along with Tina Thomas, a research fellow in our laboratory.Generate a single, patient-derived cancer cell suspension from tumor tissue using a tumor dissociation kit. Begin by transferring approximately half a gram of tumor tissue to a 100 millimeter cell culture dish containing five milliliters of enzyme supplemented RPMI buffer. Then, use scissors and tissue forceps to mince the tumor into small pieces of two to four millimeters.Pipette the tumor mixture into a dissociater tube and close the tube. Invert the tube, attach onto the sleeve of a tissue dissociater, and dissociate the tissue using the appropriate program. After dissociating, incubate the tumor cell suspension on a rotating rack at 37 degrees Celsius for one hour.Triturate the cell suspension every 15 minutes during the incubation. Following the incubation, transfer the cell suspension to a new 50 milliliter conical tube and add 10 milliliters of RPMI. Then, centrifuge the cell suspension at 314g for five minutes.After the centrifugation, remove the supernatant and suspend the pellet in five milliliters of RPMI. Pass the cell suspension through a 40 micron cell strainer and collect the strained solution into a fresh, 50 milliliter tube. After washing the strainer with five milliliters of RPMI media, centrifuge the cell suspension at 314g for five minutes to collect a pellet.Count the cells using a hemocytometer. Then, suspend the pellet in RPMI to obtain a final concentration of four times 10 to the five cells per 10 microliter volume. Transfer five microliters of this cell suspension per injection to a fresh tube, and the same volume of basement membrane matrix to make 10 microliters of cell solution for each injection and place it on ice.Transfer the mouse to the imaging platform with the abdominal side down. Fit and secure the nose cone to maintain isoflurane anesthesia. Begin the implantation procedure using hair removal lotion and a shaver to depilate the back and flank of a properly anesthetized six to eight week old immune-deficient NSG mouse.Apply optical ointment over the animal's eyes to prevent drying. Then, tape the mouse in place to prevent any inadvertent movement. Next, use ultrasound visualization to identify the murine liver, vena cava, spleen, left kidney, and adjacent left adrenal gland.Load a chilled Hamilton syringe, fitted with a small bore needle, with 10 microliters of cell solution. Then, under ultrasound guidance, gently inserted a chilled 22-gauge catheter through the skin and back muscle, directly into the left adrenal gland to provide a channel for cellular injection. Remove the needle and leave the catheter in place.Visualization of the adrenal gland throughout the insertion of the catheter, along with the needle and its trajectory, is imperative to ensure minimal injury to surrounding structures and organs and lower murine morbidity. Then, guide the syringe through the catheter positioned into the center of the adrenal gland. As the syringe is guided through the catheter, it is important to maintain catheter stability and watch the needle advance.Note that the needle itself extends approximately two millimeters beyond termination of the catheter, its position easily seen on ultrasound. Inject the cells into the targeted adrenal tissue. Leave the needle in place for one to two minutes to allow the basement membrane matrix to set.After the basement membrane matrix has set, slowly remove the needle, followed by the removal of the catheter. Finally, place the mouse into a recovery cage until it regains sufficient consciousness to maintain sternal recumbency before returning to the home cage. Ultrasound imaging monitors in vivo tumor progression.This image shows an ultrasound image of the adrenal gland, one week following injection. Here, luminescence imaging of a neuroblastoma injected mouse one week post-injection, shows readings not indicative of tumor engraftment. After two weeks, ultrasound imaging shows tumor engraftment and progression to an area of approximately 40 square millimeters.Bioluminescence at two weeks post-injection showed radiance measuring 10 to the seventh, which is suggestive of cell uptake and tumor growth correlating with ultrasound findings. Ultrasound imaging eight weeks post-injection showed continued tumor growth with an area measurement of greater than 100 square millimeters. Again, bioluminescence confirmed the ultrasound findings with increased radiance levels of 10 to the nine.3-D ultrasound imaging of the tumor showed a volume greater than 100 cubic millimeters, the baseline that our laboratory utilizes for initiation of preclinical therapeutic trials. Excised tumor grossly measured greater than one centimeter in size, correlating with ultrasound measurements and luminescence signals. This media provides demonstration of how to successfully establish adrenal orthotopic xenographs with patient-derived neuroblastoma cells, utilizing ultrasound guidance.Once mastered, this technique can be done in less than 12 minutes per injection if it is performed properly.

Research

JoVE Journal

Cancer Research

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

o<em&gt; Drosophila</em&gt; Imaginal Modelo Disc Tumor: visualização e quantificação da expressão gênica e invasão tumoral Usando Mosaicos Genéticos

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

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The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

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Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

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Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts

In vivo Imaging e Quantitation da resposta angiogênica hospedeira em xenoenxertos de tumor de zebrafish

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

Implantação ortotópica guiada por ultrassom do Adenocarcinoma Ductal Pancreático murine

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

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Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...