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施和适当的生物安全做法。核供应的小鼠严重免疫力低下, 易受环境中常见细菌引起的疾病的影响。 在准备植入和执行注射材料时, 应始终使用严格的无菌技术。
1. 细胞培养
<ol><li>生长建立的人 NB 细胞线 (SH-SY5Y, SK-N-BE2 和 IMR32) 在最小的基本培养基, 补充10% 胎牛血清, 2 毫米谷氨酰胺, 100 单位/毫升青霉素, 100 微克/毫升链霉素。补充 IMR32 细胞进一步与1毫米丙酮酸和 0.075% NaHCO3。维护37°c、5% CO2和70-80% 汇流的所有单元格.
</li>
	<li>在 RPMI 培养基中培养人类 ES 细胞系 (TC32、A673、CHLA-25 和 A4573), 辅以10% 胎牛血清和6毫米 l-谷氨酰胺。保持所有单元格在37°c, 5% CO2在70-80% 汇合.
</li>
	<li>发送所有单元格行以进行身份验证。 注: 单元格认证在外部设备上进行, 请参阅确认。</li>
</ol>2. 原发性病人源性肿瘤细胞的制备
<ol><li>在患者同意和同意下, 收获被丢弃的人的 NB 肿瘤组织从手术切除的患者为地方控制和运输到实验室在组织存贮解答保存。 注: 所有患者样本均根据机构审查委员会的指导方针被取消鉴定和处理。</li>
	<li>用肿瘤分离试剂盒从肿瘤组织中生成单个患者衍生的癌细胞悬浮液 (UMNBL001、UMNBL002)。将大约0.5 克肿瘤转移到100毫米细胞培养皿中, 含有5毫升的 RPMI 缓冲器, 200 µL 酶 H, 100 µL 酶 R 和25µL 酶 a 从人类肿瘤离解试剂盒。</li>
	<li>使用剪刀和组织钳将肿瘤切成小块 (每2-4 毫米)。将这些片段与步骤2.2 中的解决方案混合在一起。</li>
	<li>将肿瘤混合物注入 dissociator 管并关闭管。把管子倒置, 贴在组织 dissociator 的袖子上。在程序 h_tumor_01 上运行。</li>
	<li>在每15分钟间歇研磨的旋转机架上孵化37摄氏度以上的肿瘤细胞悬浮液1小时。</li>
	<li>将细胞悬浮液转移到新的50毫升圆锥管上, 加入10毫升的 RPMI。离心机细胞悬浮在 314 x g 为5分钟。</li>
	<li>取出上清液, 在5毫升的 RPMI 中悬浮颗粒。通过40µm 细胞过滤器的解决方案, 并收集到一个新鲜的50毫升圆锥管的紧张的解决方案。用5毫升的 RPMI 介质清洗此过滤器, 并将混合物在 314 x g 处用5分钟来收集颗粒。</li>
	<li>使用 hemacytometer15测量单元格密度。悬浮在 RPMI 中获得的颗粒, 以获得最终浓度为 4 x 105 cells/10 µL。采取5µL 的细胞悬浮和5µL 的胶, 使10µL 的细胞溶液的每一个注射。 注: 使用的 RPMI 量取决于细胞密度的测量。例如, 如果获得的颗粒的测量细胞密度为 4 x 105单元格, 则在 RPMI 的10µL 中悬浮颗粒。</li>
</ol>3. 细胞荧光素酶标记癌细胞
<ol><li>使10毫升的传导介质与5毫升的病毒上清 EF1a-Luc2-IRES-mCherry 和5毫升的干细胞培养基。添加7.5 µL 1x 凝聚胺。</li>
	<li>在转导介质中混合 5 x 106癌细胞, 在100毫米超低附着板中加入平板。在37摄氏度和 5% CO2上孵化过夜。 注: 当使用低附着板时, 细胞不会粘附在板上。</li>
	<li>24小时后, 将含有细胞的100毫米板置于组织培养罩下, 将混合物转化为15毫升锥形管。离心机的细胞悬浮在 314 x g 5 分钟得到细胞颗粒。用1毫升的1x 磷酸盐缓冲盐水 (PBS) 清洗细胞颗粒, 并在1毫升的 HBSS 中悬浮细胞颗粒, 其中含有1% 胎牛血清 (血清)。<ol>
<li>基于 GFP-阳性信号对荧光活化细胞分选 (资产管制) 分析进行分类。将排列的细胞在100毫米组织培养处理皿上, 在70-80% 汇流、37°c 和 5% CO2之前保持, 直到需要为止。</li>
	</ol>
</li>
	<li>用荧光素酶检测试剂盒确认荧光素酶信号。板 10 x 104在一个照度计兼容96井板和孵化细胞一夜地在37°c 和 5%CO2 的每井被排序的细胞。24小时后, 将96井板带入室温, 并在1:1 配比的水井中加入稳定格洛试剂。允许细胞溶解5分钟, 并在分光光度计中读出发光。</li>
	<li>把 100 mm 的盘子放在组织培养罩下。丢弃上清介质。用2毫升 1x PBS 清洗细胞一次。</li>
	<li>添加2毫升0.25% 胰蛋白酶, 并返回100毫米菜的孵化器为2-3 分钟。2-3 分钟后, 检查脱落细胞显微镜下, 并添加8毫升的 RPMI 停用胰蛋白酶。</li>
	<li>收集细胞悬浮在15毫升圆锥管和离心机在 314 x g 5 分钟得到一个球团。放弃上清。用1毫升的 1x PBS 清洗细胞颗粒, 将颗粒悬浮在5毫升的 RPMI 中, 并使用 hemacytometer15确定细胞密度。离心机 yhe 剩余细胞溶液在 314 x g 5 分钟获得颗粒。</li>
	<li>在 RPMI 中暂停颗粒, 以获得浓度为 4 x 105 cells/10 µL。采取5µL 的细胞悬浮和5µL 的胶, 使10µL 的细胞溶液的每一个注射。</li>
</ol>4. 超声引导肾上腺 (NB) 或肾亚胶囊 (ES) 植入术
注意: 所有超声过程都是使用高分辨率的体内成像系统进行的。为被描述的规程, 传感器, 有40兆赫的中心频率和22-55 兆赫带宽使用了。
<ol><li>在所有注射过程中利用6-8 周的免疫缺陷小鼠。</li>
	<li>冷胶和蒸压哈密尔顿注射器装有27G 针 (1.25), 和22G 导管 (0.9 毫米外径, 25 毫米长度) 隔夜在4摄氏度。</li>
	<li>将在步骤3中准备的单元解决方案放在冰上。</li>
	<li>麻醉使用2% 异氟醚在 O2中的诱导腔内的小鼠, 以2升/分的交货。 注: 充分的麻醉是由于缺乏主动运动和维持自发性呼吸而决定的。</li>
	<li>一旦麻醉, 脱毛的背部和侧面的动物使用脱毛乳液 (如奈尔) 和剃须刀。</li>
	<li>将动物转移到腹部侧面的成像平台上, 将动物的鼻子放到 nosecone 中, 并在吸入异氟醚时得到保护。</li>
	<li>将光学软膏放在动物的眼睛里以防止干燥。将鼠标贴在原处以防止任何无意的移动。</li>
	<li>应用超声可视化法识别小鼠肝脏、腔静脉、脾、左肾及相邻的左肾上腺。</li>
	<li>使用无菌手套, 口罩和帽子的个人保护和维护的无菌条件。在超声引导下, 轻轻地插入一个22G 导管通过皮肤和背部肌肉直接进入左肾上腺, 提供一个通道的细胞注射。取出针头, 把导管放到位。</li>
	<li>装上装有10µL 细胞溶液的小口径针头的冷冻哈密尔顿注射器, 然后通过导管定位到肾上腺中心, 引导注射器 stereotactically。</li>
	<li>将细胞注入靶向肾上腺组织。把针放到位1-2 分钟, 让胶设置。慢慢取出针头, 然后取出导管。</li>
	<li>把老鼠放回笼子里, 确保老鼠恢复足够的意识来维持胸骨卧床。 注意: 由于这种技术的微创性质, 术后疼痛是极小的, 但仍然监测每天的基础上与小鼠的健康检查. "应该跟着一个句子:" 如果在课程中发现意外的疼痛或窘迫的迹象在一项实验中, 请咨询您的机构兽医支持单位, 以了解有关镇痛或其他医疗护理的信息。 
</li>
</ol>

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作者没有披露。

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 ...

utilization-ultrasound-guided-tissue-directed-cellular-implantation

Research

JoVE Journal

Cancer Research

8.2K 次观看.  The University of Michigan Medical School. 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。

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

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

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

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

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

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

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. However, little is known about the heterogeneity of liver metastases, and animal models capable of evaluating the development of individual metastatic colonies are needed. Here, we present an advanced model of hepatic metastases that provides the ability to quantitatively visualize the development of individual tumor clones in the liver and estimate their growth kinetics and colonization efficiency. We generated a panel of monoclonal derivatives of HCT116 human colorectal cancer cells stably labeled with luciferase and tdTomato and possessing different growth properties. With a splenic injection followed by a splenectomy, the majority of these clones are able to generate hepatic metastases, but with different frequencies of colonization and varying growth rates. Using the In Vivo&#160;Imaging System (IVIS), it is possible to visualize and quantify metastasis development with in vivo luminescent and ex vivo fluorescent imaging. In addition, Diffuse Luminescent Imaging Tomography (DLIT) provides a 3D distribution of liver metastases in vivo. Ex vivo fluorescent imaging of harvested livers provides quantitative measurements of individual hepatic metastatic colonies, allowing for the evaluation of the frequency of liver colonization and the growth kinetics of metastases. Since the model is similar to clinically observed liver metastases, it can serve as a modality for detecting genes associated with liver metastasis and for testing potential ablative or adjuvant treatments for liver metastatic disease.

The ability of metastatic clones to colonize distant sites depends on their proliferation capacity and/or their ability to survive in the host microenvironment without significant proliferation. Here, we present an animal model that allows quantitative visualization of both types of liver colonization by 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

Isolated hilar and mediastinal recurrence (IMHR) following external beam radiation therapy (EBRT) in patients with lung cancer is common. These patients do not have many treatment options and are usually offered palliative chemotherapy or best supportive care. Endobronchial ultrasound (EBUS)-guided intratumoral injection of cisplatin (ITC) is a novel approach for these patients. The procedure is performed under conscious sedation. The lesion is located with a bronchoscopy using EBUS, and a 22-gauge EBUS needle is advanced through the working channel of the scope and locked in position. Under ultrasound guidance, the wall of the tracheobronchial tree is punctured and the needle is moved into the target lesion. The needle stylet is then removed and cisplatin (40 mg/40 mL) is injected into the lesion. One to two sites are treated per session. Details of the procedure are described in the protocol section of paper. At our center, 50 sites were treated in 36 patients (19 males, 17 females). The mean age of our cohort was 61.9 &#177;8.5 years. We performed final analyses on 35 patients and 41 sites. 24/35 (69%) had complete or partial response (responders), whereas 11/35 (31%) had stable or progressive disease (non-responders). Overall, survival in our group was 8 months (95% CI of 6-11 months), with patients who responded having significantly better survival than the ones who did not.

The management of isolated recurrent lung cancer in a previously-irradiated field is challenging. Here, we describe an endobronchial ultrasound (EBUS)-guided cisplatin injection for the management of patients with localized lung cancer recurrence in a previously-radiated field.

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 ...

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

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

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 ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

数字聚合酶链反应法测定零星家族性腺瘤性息肉病患者的基因变异

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

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

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

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

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 zebrafish xenograft is created by implanting mammalian tumor cells into the perivitelline space of two days-post-fertilization zebrafish embryos, followed by measuring the extent of the angiogenic response observed at an experimental endpoint up to two days post-implantation. The key advantage to this method is the ability to accurately quantitate the zebrafish host angiogenic response to the graft. This enables detailed examination of the molecular mechanisms as well as the host vs tumor contribution to the angiogenic response. The xenografted embryos can be subjected to a variety of treatments, such as incubation with potential anti-angiogenesis drugs, in order to investigate strategies to inhibit tumor angiogenesis. The angiogenic response can also be live-imaged in order to examine more dynamic cellular processes. The relatively undemanding experimental technique, cheap maintenance costs of zebrafish and short experimental timeline make this model especially useful for the development of strategies to manipulate tumor angiogenesis.

The aim of this method is to generate an in vivo model of tumor angiogenesis by xenografting mammalian tumor cells into a zebrafish embryo that has fluorescently-labelled blood vessels. By imaging the xenograft and associated vessels, a quantitative measurement of the angiogenic response can be obtained.

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

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

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

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

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 ...

The Establishment and Utilization of Patient Derived Xenograft Models of Central Nervous System Metastasis

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately represent the disease. Patient derived xenograft (PDX) models of CNS metastasis have been shown to better represent the phenotypic and molecular characteristics of the human disease, as well as better reflect the heterogeneity and clonal dynamics of human patient tumors compared to historic cell line models. There are multiple sites that can be used to implant patient-derived tissue when setting up preclinical trials, each with their own advantages and disadvantages, and each suited for studying different aspects of the metastatic cascade. Here, the protocol describes how to establish PDX models and present three different approaches for utilizing CNS metastasis PDX models in pre-clinical studies, discussing each of their applications and limitations. These include flank implantation, orthotopic injection in the brain, and intracardiac injection. Subcutaneous flank implantation is the easiest to monitor and, therefore, most convenient for pre-clinical studies. In addition, metastases to brain and other tissues from flank implantation were observed, indicating that the tumor has undergone multiple steps of metastasis, including intravasation, extravasation, and colonization. Orthotopic injection in the brain is the best option for recapitulating the brain tumor microenvironment and is useful for determining the efficacy of biologics to cross the blood-brain barrier (BBB) but bypasses most steps of the metastatic cascade. Intracardiac injection facilitates metastasis to the brain and is also useful for studying organ tropism. While this method forgoes earlier steps of the metastatic cascade, these cells will still have to survive circulation, extravasate, and colonize. The utility of a PDX model, is therefore, impacted by the route of tumor inoculation and the choice of which one to utilize should be dictated by the scientific question and overall goals of the experiment.

Central nervous system metastasis PDX models represent the phenotypic and molecular characteristics of human metastasis, making them excellent models for preclinical studies. Described here is how to establish PDX models and the inoculation routes that are best used for preclinical studies.