Name: utilization of ultrasound guided tissue-directed cellular implantation for the establishment of biologically relevant metastatic tumor xenografts
Uploaded: 2018-05-25T14:30:00.000+00:00
Duration: 7 min 50 s
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.
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	<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><tbody><tr><td>&lt;strong&gt;Mice&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>NOD-SCID</td><td>Charles River</td><td>394</td><td></td></tr><tr><td>NSG</td><td>The Jackson Laboratory</td><td>5557</td><td></td></tr><tr><td>&lt;strong&gt;Cell Line&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;NB&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>IMR-32</td><td>ATCC</td><td>CCL-127</td><td>Established human neuroblastoma cell line</td></tr><tr><td>SH-SY5Y</td><td>ATCC</td><td>CRL-2266</td><td>Established human neuroblastoma cell line</td></tr><tr><td>SK-N-Be2</td><td>ATCC</td><td>CRL-2271</td><td>Established human neuroblastoma cell line</td></tr><tr><td>&lt;strong&gt;ES&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>TC32&amp;nbsp;</td><td>COGcell.ORG</td><td></td><td>Established human Ewing&#039;s Sarcoma cell line</td></tr><tr><td>A673</td><td>COGcell.ORG</td><td></td><td>Established human Ewing&#039;s Sarcoma cell line</td></tr><tr><td>CHLA-25</td><td>COGcell.ORG</td><td></td><td>Established human Ewing&#039;s Sarcoma cell line</td></tr><tr><td>A4573</td><td>COGcell.ORG</td><td></td><td>Established human Ewing&#039;s Sarcoma cell line</td></tr><tr><td>&lt;strong&gt;Cell Line media&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>RPMI</td><td>Life Technologies</td><td>11875-093</td><td></td></tr><tr><td>Matrigel</td><td>BD BioSciences</td><td>354234</td><td></td></tr><tr><td>&lt;strong&gt;Dissociation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dissection Tools</td><td>KentScientific</td><td>INSMOUSEKIT</td><td></td></tr><tr><td>Human Tumor Dissociation Kit&amp;nbsp;</td><td>MACS Miltenyi Biotec</td><td>130-095-929</td><td></td></tr><tr><td>gentleMACS dissociator</td><td>MACS Miltenyi Biotec</td><td>130-093-235</td><td></td></tr><tr><td>gentleMACS C tubes</td><td>MACS Miltenyi Biotec</td><td>130-096-334</td><td></td></tr><tr><td>Cell Strainer</td><td>Corning</td><td>431751</td><td></td></tr><tr><td>&lt;strong&gt;Luciferase Tagging&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Lenti-GFP1 virus</td><td>University of Michigan, Vector Core</td><td></td><td>Luciferase Virus</td></tr><tr><td>Steady Glo-Luciferase Assay Kit</td><td>Promega</td><td>E2510</td><td></td></tr><tr><td>&lt;strong&gt;Bioluminescence Imaging&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ivis Spectrum Imaging System</td><td>PerkinElmer</td><td>124262</td><td></td></tr><tr><td>D-Luciferin</td><td>Promega</td><td>E160X</td><td></td></tr><tr><td>&lt;strong&gt;Anesthetic&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Inhaled Isoflurane&amp;nbsp;</td><td>Piramal Critical Care Inc</td><td>66794-0017-25</td><td></td></tr><tr><td>&lt;strong&gt;Ultrasound Guided Injection&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Vevo 2100 High Resolution Imaging</td><td>Vevo</td><td>2100</td><td></td></tr><tr><td>Hamilton Syringes (27 gauge needle)</td><td>Hamilton</td><td>80000</td><td></td></tr><tr><td>22 Gauge Angiocatheter</td><td>BD Biosciences</td><td>381423</td><td></td></tr><tr><td>Optical ointment</td><td>Major Pharmaceuticals</td><td>301909</td><td></td></tr><tr><td>Nair</td><td>Church &amp;amp; Dwight Co</td><td></td><td>Hair Removal agent</td></tr><tr><td>Aquasonic 100 Ultrasound Transmission gel</td><td>Parker</td><td></td><td>Ultrasound gel</td></tr><tr><td>&lt;strong&gt;Histology&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CD99</td><td>DAKO</td><td>M3601</td><td>Primary Antibody</td></tr><tr><td>Tyrosine Hydroxylase</td><td>Sigma-Aldrich</td><td>T2928</td><td>Primary Antibody</td></tr><tr><td>Secondary HRP-Polymer antibody</td><td>Biocare</td><td>BRR4056KG</td><td></td></tr><tr><td>&lt;strong&gt;Miscelleneous&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10 mL Pipettes</td><td>Fisher Scientific</td><td>13-676-10J</td><td></td></tr><tr><td>5 mL Pipettes</td><td>Fisher Scientific</td><td>13-676-10H</td><td></td></tr><tr><td>1.5 mL Microcentrifuge tubes</td><td>Fisher Scientific</td><td>05-408-129</td><td></td></tr><tr><td>P1000 pipette</td><td>Eppendorf</td><td>3120000062</td><td></td></tr><tr><td>P200 pipette</td><td>Eppendorf</td><td>3120000054</td><td></td></tr><tr><td>P1000 pipette tips</td><td>Fisher Scientific</td><td>21-375E</td><td></td></tr><tr><td>P200 pipette tips</td><td>Fisher Scientific</td><td>21-375D</td><td></td></tr><tr><td>Portable pipette aid</td><td>Drummond</td><td>4-000-101</td><td></td></tr><tr><td>digital animal Weighing Scale&amp;nbsp;</td><td>KentScientific</td><td>SCL-1015</td><td></td></tr><tr><td>Calipers</td><td>Fisher Scientific</td><td>06-664-16</td><td></td></tr><tr><td>6well low attachment plates</td><td>Corning</td><td>07-200-601</td><td></td></tr><tr><td>10 cm Tissue Culture Treated Dishes</td><td>Fisher Scientific</td><td>FB012924</td><td></td></tr><tr><td>Polybrene</td><td>Sigma-Aldrich</td><td>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

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

患者衍生的人类泌尿细胞癌和结肠直肠癌肿瘤生长和自发转移的正交异种移植模型

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

科研

癌症研究

Generation of a Liver Orthotopic Human Uveal Melanoma Xenograft Platform in Immunodeficient Mice

In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more representative models to study human cancers in immunodeficient mice than traditional established human cell lines in vitro. Recently, orthotopically implanted patient-derived tumor xenograft (PDX) models in mice have been developed to better replicate features of patient tumors. A liver orthotopic xenograft mouse model is expected to be a useful cancer research platform, providing insights into tumor biology and drug therapy. However, liver orthotopic tumor implantation is generally complicated. Here we describe our protocols for the orthotopic implantation of patient-derived liver-metastatic uveal melanoma tumors. We cultured human liver metastatic uveal melanoma cell lines into immunodeficient mice. The protocols can result in consistently high technical success rates using either a surgical orthotopic implantation technique with chunks of patient-derived uveal melanoma tumor or a needle injection technique with cultured human cell line. We also describe protocols for CT scanning to detect interior liver tumors and for re-implantation techniques using cryopreserved tumors to achieve re-engraftment. Together, these protocols provide a better platform for liver orthotopic tumor mouse models of liver metastatic uveal melanoma in translational research.

Orthotopic human liver metastatic uveal melanoma xenograft mouse models were created using surgical orthotopic implantation techniques with patient-derived tumor chunk and needle injection techniques with cultured human uveal melanoma cell lines.

在免疫缺陷小鼠中生成肝脏正交人子宫黑色素瘤异种移植平台

In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more ...

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 ...

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic target analyses and drug response monitoring using patient derived xenografts (PDX). This strategy was validated using ovarian tumors as an example. The mate-pair next generation sequencing (MPseq) protocol was used to identify structural alterations and followed by analysis of potentially targetable alterations. Human tumors grown in immunocompromised mice were treated with drugs selected based on the genomic analyses. Results demonstrated a good correlation between the predicted and the observed responses in the PDX model. The presented approach can be used to test the efficacy of combination treatments and aid personalized treatment for patients with recurrent cancer, specifically in cases when standard therapy fails and there is a need to use drugs off label.

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients&#8217; tumors into mice and testing responses to corresponding drugs.

使用结构DNA改变分析和患者衍生异种移植测试癌症的靶向疗法

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These lesions compromise bone structure, increase the risk of pathologic fracture, and leave patients with limited treatment options. Primary bone tumors metastasize to distant organs, with some types capable of spreading to other skeletal sites. However, recent evidence suggests that with many solid tumors, cancer cells that have spread to bone may be the primary source of cells that ultimately metastasize to other organ systems. Most syngeneic or xenograft mouse models of primary bone tumors involve intra-osseous (orthotopic) injection of tumor cell suspensions. Some animal models of skeletal metastasis from solid tumors also depend on direct bone injection, while others attempt to recapitulate additional steps of the bone metastatic cascade by injecting cells intravascularly or into the organ of the primary tumor. However, none of these models develop bone metastasis reliably or with an incidence of 100%. In addition, direct intra-osseous injection of tumor cells has been shown to be associated with potential tumor embolization of the lung. These embolic tumor cells engraft but do not recapitulate the metastatic cascade. We reported a mouse model of osteosarcoma in which fresh or cryopreserved tumor fragments (consisting of tumor cells plus stroma) are implanted directly into the proximal tibia using a minimally invasive surgical technique. These animals developed reproducible engraftment, growth, and, over time, osteolysis and lung metastasis. This technique has the versatility to be used to model solid tumor bone metastasis and can readily employ grafts consisting of one or multiple cell types, genetically-modified cells, patient-derived xenografts, and/or labeled cells that can be tracked by optical or advanced imaging. Here, we demonstrate this technique, modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone.

Bone metastasis models do not develop metastasis uniformly or with a 100% incidence. Direct intra-osseous tumor cell injection can result in embolization of the lung. We present our technique modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone, leading to reproducible engraftment and growth.

通过实体瘤移植植入骨模拟原发性骨肿瘤和骨转移

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

皮下及肝内人肝癌裸鼠移植瘤的免疫缺陷小鼠的产生

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

医学

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.

患者衍生的中枢神经系统转移异种移植模型的建立与利用

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately ...

Generation of an Orthotopic Xenograft of Pancreatic Cancer Cells by Ultrasound-Guided Injection

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant behavior and high resistance to therapeutic treatments. Indeed, despite many efforts, both standard chemotherapy and innovative target therapies have substantially failed when moved from preclinical evaluation to the clinical setting. In this scenario, the development of preclinical mouse models better mimicking in vivo characteristics of PCa is urgently needed to test newly developed drugs. The present protocol describes a method to generate a mouse model of PCa, represented by an orthotopic xenograft obtained by ultrasound-guided injection of human pancreatic tumor cells. Using such a reliable and minimally invasive protocol, we also provide evidence of in vivo engraftment and development of tumor masses, which can be monitored by ultrasound (US) imaging. A noteworthy aspect of the PCa model described here is the slow development of the tumor masses over time, which allows precise identification of the starting point for pharmacological treatments and better monitoring of the effects of therapeutic interventions. Moreover, the technique described here is an example of implementation of the 3Rs principles since it minimizes pain and suffering and directly improves the welfare of animals in research.

We present a protocol to generate a minimally invasive orthotopic pancreatic cancer model by ultrasound-guided injection of human pancreatic cancer cells and the subsequent monitoring of tumor growth in vivo by ultrasound imaging.

通过超声引导注射产生胰腺癌细胞的原位异种移植物

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

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

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

微创建立小鼠原位膀胱移植瘤的

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

<meta charset="utf8"></head><body>乳腺癌患者来源的异种移植物的高通量解离和原位植入

High-Throughput Dissociation and Orthotopic Implantation of Breast Cancer Patient-Derived Xenografts

Patient-derived xenografts (PDXs) provide a clinically relevant method for recapitulating tumor-involved cell types and the tumor microenvironment, which is essential for advancing knowledge of breast cancer (BC). Additionally, PDX models enable the study of BC systemic effects, which is not possible using in vitro models. Traditional methods for implanting BC xenografts typically involve anesthesia and sterile surgical procedures, which are time-consuming, invasive, and limit the scalability of PDX models in BC research. This protocol describes a simple and scalable method for the orthotopic implantation of BC PDXs in mice. The immunodeficient mouse strain NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) was used for PDX engraftment. Human BC samples obtained from IRB-consented patients were mechanically and enzymatically dissociated, then resuspended in a solution of basement membrane extract (BME) and RPMI 1640. Animals were restrained by scruffing, and depilatory cream was applied to remove hair from the fat pads at the fourth inguinal nipple, followed by injection. Approximately 2 million cells in a 100 &#181;L suspension were bilaterally injected orthotopically into the mammary fat pads using a 26 G needle. Notably, no anesthetic was required, and the total procedure time was under 5 min, from cell preparation to injection. After a growth period of several months, tumors were excised and processed for authentication. Validation included receptor status assessment using immunohistochemistry with specific antibodies for traditional BC receptors (i.e., ER, PR, HER2). Tumor morphology was confirmed with hematoxylin and eosin (H&amp;E) staining, which was interpreted by a pathologist. Genetic similarity to the patient sample was verified through bulk RNA sequencing and short tandem repeat (STR) analysis. This approach to PDX engraftment and validation supports the rigorous development of models and high-throughput tumor implantation, enabling well-powered studies across various BC subtypes.

This protocol describes an efficient, non-surgical method for the orthotopic implantation of breast cancer patient-derived xenografts in mice. The technique involves enzymatic tumor dissociation followed by direct injection into the mammary fat pads, enabling high-throughput implantation. Comprehensive validation ensures model fidelity, facilitating rigorous studies across various breast cancer subtypes.

乳腺癌患者来源的异种移植物的高通量解离和原位植入

Patient-derived xenografts (PDXs) provide a clinically relevant method for recapitulating tumor-involved cell types and the tumor microenvironment, which ...