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

这项工作得到了罗伯特伍德约翰逊基金会/阿莫斯医学教师发展计划, Taubman 研究所, 和美国密歇根大学儿科手术科的支持。作者希望感谢金柏 Converso-巴兰和马库斯博士 Jarboe 为协助与超声注射程序和成像平台。我们感谢保罗. 特朗布利为图图形提供的帮助。我们还感谢密歇根大学放射科使用的分子影像学中心和肿瘤影像核, 这部分是由综合癌症中心 NIH (P30 CA046592) 支持的。密歇根大学生理学分型核心部分由 NIH (OD016502) 和法兰克心血管中心资助。IDEXX RADIL 生物学研究设施, 哥伦比亚, 密苏里州进行了细胞线认证。我们感谢苔米斯托尔, Rajen 博士和莫特固体肿瘤肿瘤学项目。我们的病人和家人感谢他们的启发, 勇气和持续的支持我们的研究。

所有工作都是按照密歇根大学机构审查委员会 (嗡嗡声 00052430) 进行的, 符合大学动物使用和照料委员会 (UCUCA) 批准的程序。动物医学实验室 (乌拉姆) 的单位监督动物护理。
所有工作都是在密歇根大学机构审查委员会 (嗡嗡声 00052430) 的批准下完成的, 符合所有人类研究伦理委员会的规定。人类细胞被认为是一种潜在的 biohazardous 材料, 因此请遵循您的机构所需的所有特殊预防措...

<ol>
	<li>Sanmamed, M. F., Chester, C., Melero, I., Kohrt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+optimal+murine+models+to+investigate+immune+checkpoint+blockers+and+their+combination+with+other+immunotherapies.">Defining the optimal murine models to investigate immune checkpoint blockers and their combination with other immunotherapies.</a> Ann Oncol. 27 (7), 1190-1198 (2016).</li><li>Fidler, I. J., Hart, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+diversity+in+metastatic+neoplasms:+origins+and+implications.">Biological diversity in metastatic neoplasms: origins and implications.</a> Science. 217 (4564), 998-1003 (1982).</li><li>Bibby, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+models+of+cancer+for+preclinical+drug+evaluation.">Orthotopic models of cancer for preclinical drug evaluation.</a> Eur J Cancer. 40 (6), 852-857 (2004).</li><li>Killion, J. J., Radinsky, R., Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+Models+are+Necessary+to+Predict+Therapy+of+Transplantable+Tumors+in+Mice.">Orthotopic Models are Necessary to Predict Therapy of Transplantable Tumors in Mice.</a> Cancer Metastasis Rev. 17 (3), 279-284 (1998).</li><li>Daniel, V. C., 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=A+primary+xenograft+model+of+small-cell+lung+cancer+reveals+irreversible+changes+in+gene+expression+imposed+by+culture+in+vitro.">A primary xenograft model of small-cell lung cancer reveals irreversible changes in gene expression imposed by culture in vitro.</a> Cancer Res. 69 (8), 3364-3373 (2009).</li><li>Khanna, C., Jaboin, J. J., Drakos, E., Tsokos, M., Thiele, C. 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. Vis. Exp. (84), e51123 (2014).</li><li>Teitz, T., 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=Preclinical+Models+for+Neuroblastoma:+Establishing+a+Baseline+for+Treatment.">Preclinical Models for Neuroblastoma: Establishing a Baseline for Treatment.</a> PLoS ONE. 6 (4), e19133 (2011).</li><li>Braekeveldt, N., 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=Neuroblastoma+patient-derived+orthotopic+xenografts+retain+metastatic+patterns+and+geno-+and+phenotypes+of+patient+tumours.">Neuroblastoma patient-derived orthotopic xenografts retain metastatic patterns and geno- and phenotypes of patient tumours.</a> International Journal of Cancer. 136 (5), 252-261 (2015).</li><li>Van Noord, R. A., 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=Tissue-directed+Implantation+Using+Ultrasound+Visualization+for+Development+of+Biologically+Relevant+Metastatic+Tumor+Xenografts.">Tissue-directed Implantation Using Ultrasound Visualization for Development of Biologically Relevant Metastatic Tumor Xenografts.</a> In Vivo. 31 (5), 779-791 (2017).</li><li>Vormoor, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Preclinical+Orthotopic+Xenograft+Model+of+Ewing+Sarcoma+and+Other+Human+Malignant+Bone+Disease+Using+Advanced+In+Vivo+Imaging.">Development of a Preclinical Orthotopic Xenograft Model of Ewing Sarcoma and Other Human Malignant Bone Disease Using Advanced In Vivo Imaging.</a> PLoS ONE. 9 (1), e85128 (2014).</li><li>Hakky, T. S., Gonzalvo, A. A., Lockhart, J. L., Rodriguez, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+Ewing+sarcoma+of+the+kidney:+a+symptomatic+presentation+and+review+of+the+literature.">Primary Ewing sarcoma of the kidney: a symptomatic presentation and review of the literature.</a> Ther Adv Urol. 5 (3), 153-159 (2013).</li><li>Cheng, H., Clarkson, P. W., Gao, D., Pacheco, M., Wang, Y., Nielsen, T. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+Antibodies+Targeting+CSF1+Impede+Macrophage+Recruitment+in+a+Xenograft+Model+of+Tenosynovial+Giant+Cell+Tumor.">Therapeutic Antibodies Targeting CSF1 Impede Macrophage Recruitment in a Xenograft Model of Tenosynovial Giant Cell Tumor.</a> Sarcoma. 2010, 174528 (2010).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+a+Hemacytometer+to+Count+Cells.">Using a Hemacytometer to Count Cells.</a> Basic Methods in Cellular and Molecular Biology. , (2018).</li></ol>

超声引导下的铌和 ES 细胞植入术是建立可靠的小鼠移植物在肿瘤生物学基础研究中的有效和安全的方法。对超声引导组织靶向植入的成功至关重要的是, 训练有素的人员在解剖性地定位了感兴趣的器官和在立体定向注射肿瘤细胞方面具有专门知识。
肿瘤组织的分离被证明是发展描述的患者衍生异体移植模型的关键步骤。原生肿瘤组织包括细胞微环境和周围基质, 可能会影响肿...

动物移植模型是新的抗癌治疗的前期研究的基本工具。标准的小鼠移植依赖于皮下侧植入细胞, 为监测肿瘤的生长提供了一个有效的、易于访问的场所。皮下模型的缺点是缺乏肿瘤特异性的生物学特性, 这可能限制其转移1的潜能。这种限制是通过使用同种异体原位移植来克服的, 肿瘤细胞被嫁接在当地的组织部位, 提供一个相关的微环境与转移电位2。同种异体原位移植模型保持原有的生物学特性, 为临床前药物发现提供可靠的模型3,4。用于组织定向植入的癌细胞要么是建立的细胞系, 要么是病人肿瘤的患者源细胞。从癌细胞系建立的异位移植可能表现出与原发性肿瘤的遗传差异很大, 与患者派生的移植性5相比。鉴于此, 建立患者源性同种异体原位移植已成为检测肿瘤药物发现新疗法的首选标准。
在儿科癌神经母细胞瘤 (nb), 原位移植模型重述原发肿瘤生物学和发展转移到典型的站点的 NB 传播6,7。NB 发育在肾上腺或沿旁交感神经链。最常见的原位植入方法需要开放的跨腹部手术程序。这种方法往往繁琐, 动物发病率高, 恢复期复杂。高分辨率超声最近被用于组织定向植入肿瘤细胞的发展, 几个小鼠模型的癌症研究8,9。该技术是可靠的, 可重复的, 高效的, 并安全的建立相关的转移瘤移植10,11。
以超声引导靶器官定位和针植入细胞系和患者源性肿瘤细胞为例, 建立儿童肿瘤移植术11。该技术用于小鼠肾上腺的 NB 靶向。尤文的肉瘤主要是一种骨癌, 常见于长骨, 如股骨和骨盆骨12。病例报告表明, 为了确定一个主要的骨癌的生长是否可行的肾脏组织, 一个肾亚囊的位置被选择为原位植入13。肿瘤细胞的肾小球囊细胞植入术是研究 ES14自发转移的一个有希望的模型。

<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

抗癌疗法的临床前试验依赖于类似于癌症先天倾向的异种异体模型。标准皮下侧面模型的优点包括程序性缓解和监测肿瘤进展和反应的能力, 没有侵袭性成像。这种模型往往是不一致的转化临床试验, 并具有有限的生物学相关特性与低倾向于产生转移, 因为缺乏一个本土的微环境。相比之下, 原生肿瘤部位的同种异体原位移植模型可以模拟肿瘤微环境, 复制远处转移扩散等重要疾病特征。这些模型往往需要繁琐的手术程序, 延长麻醉时间和恢复期。为了解决这一问题, 癌症研究人员最近利用超声引导注射技术建立了用于临床前实验的癌症异种模型, 从而可以快速可靠地建立组织导向的小鼠模型。超声可视化也为肿瘤植入和生长的纵向评估提供了一种无创的方法。在这里, 我们描述了超声引导下注射癌细胞的方法, 利用肾上腺的 NB 和肾亚胶囊的 ES。这种微创方法克服了在组织特定部位的癌细胞在生长和转移中的繁琐开放性手术植入, 并消退了病态恢复期。我们描述了已建立的细胞系和病人的细胞系在原位注射的应用。预先制作的商业套件可用于肿瘤离解和荧光素酶标记细胞。用图像引导注射细胞悬浮液为临床前模型的建立提供了一个微创和可再生的平台。该方法用于为其他癌症 (如膀胱、肝脏和胰腺) 建立可靠的前置模型, 以此为许多癌症模型的未开发潜力提供依据。

使用所提出的程序, 超声引导的铌细胞植入肾上腺是在一个专门的程序室, 配备了一个加热手术表。手臂和脚垫被放置监测鼠心脏活动 (图 1A)。用鼻锥吸入法在异氟醚下麻醉动物。使用高分辨率超声探头, 左肾被发现与肾上腺刚刚头骨的肾脏 (图 1B)。针在直接可视化 (图 1C) 下进入肾上腺。在该技术的初步应...

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

在这里, 我们提出了一个协议, 利用超声引导注射神经母细胞瘤 (NB) 和尤文的肉瘤 (ES) 干细胞 (已建立的细胞系和病人派生的肿瘤细胞) 在生物相关的网站, 以创建可靠的前模型癌症研究。

超声引导组织定向细胞植入术在生物学相关转移性肿瘤移植中的应用

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

该<em&gt;果蝇</em&gt;成虫盘瘤模型：基因的表达与肿瘤侵袭性的可视化和定量遗传马赛克

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

科研

癌症研究

Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones

肝大肠癌转移的高级动物模型：成像技术和转移性克隆性质

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. ...

Endobronchial Ultrasound-guided Intratumoral Injection of Cisplatin for the Treatment of Isolated Mediastinal Recurrence of Lung Cancer

支气管内超声引导下顺铂瘤内注射对肺癌纵隔孤立复发的治疗

Isolated hilar and mediastinal recurrence (IMHR) following external beam radiation therapy (EBRT) in patients with lung cancer is common. These patients ...

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

可追踪记者乳腺癌患者来源的移植瘤贴标肿瘤生长和转移的研究

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

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

肺定植法在肺部组织循环肿瘤细胞可视化中的建立

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

循环肿瘤细胞的微操作, 用于下游分子分析和转移电位评估

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

在斑马鱼肿瘤异种移植物中宿主血管反应的Vivo成像和定量

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

毛尿胰腺腺腺腺癌超声引导正射植入

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

斑马鱼原发性患者衍生肿瘤异种移植物的药物筛选

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