Name: implantation and monitoring by pet/ct of an orthotopic model of human pleural mesothelioma in athymic mice
Uploaded: 2019-12-21T10:00:00
Description: Watch this Scientific Journal Video about Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice at JoVE.com

这项研究由利格·吉纳沃塞癌症（V.S.-B.）和日内瓦和洛桑大学和医院的生物医学成像中心（CIBM）资助（到D.J.C.、O.B.和S.G.）。

下文描述的所有程序均经机构动物护理和使用委员会以及瑞士日内瓦兽医国家办公室（授权 GE/106/16）批准。MPM细胞系H2052/484在我们的实验室建立和特征，详细在科林DJ等人的文章23。简单地说，H2052/484细胞系是从将NCI-H2052（ATCC）细胞注射到免疫缺陷裸鼠体内后获得的胸腔肿瘤建立的。
1. 实验设计
<ol><li>使用统计功率计算（例如<a href="http://powerandsamplesize.com/...

<ol>
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本文介绍了注射在贫血小鼠胸腔中的MPM H2052/484细胞的原始正交模型，以及一种通过小动物PET/CT成像进行监测的方法。该模型可以实施与适度的动物处理和手术技能，并显示一个非常好的发展速度。它允许在未治疗的小鼠中，在未治疗的小鼠中留出约10周的大实验窗口，并在注射后2周内对肿瘤进行非侵入性纵向检测。
正交模型依赖于将活细胞或组织直接植入肿瘤的初始环境中?...

恶性胸膜内皮瘤（MPM）是一种癌症，最常见的是与接触石棉纤维1，2，3。虽然石棉在大多数西方国家已被禁用4，5，6，MPM的发病率仍然增加7，8。最近，老鼠接触碳纳米管表明，它们可能导致人类健康风险很大。数据表明，接触这些产品可能导致慢性炎症和分子变化（例如，肿瘤抑制途径的丧失），从而导致恶性间皮瘤的恶化。目前，多壁碳纳米管是纳米技术最重要的产品之一，并越来越多地被纳入各种产品，如复合材料、储能材料、医药、电子和环境修复材料。
MPM是一种预后不佳的癌症，大多数患者在诊断后两年内死亡，因为目前治疗方式11的疗效有限。MPM治疗的选择取决于癌症阶段。对于大多数早期MPM（阶段1，可能一些阶段2或3肿瘤），临床方法是一种多模态疗法，包括肿瘤的手术切除，与放疗和化疗12相关。结合顺铂和pemxed联合化疗，用于治疗大多数被诊断为晚期局部侵入性疾病的患者，即不适合手术切除，或否则不适合治疗手术13，14。因此，迫切需要为MPM患者开发更有效的治疗方法。然而，很少有经过验证的体内动物模型能够反映MPM的临床相关性。几个小鼠MPM模型已经开发，但其中大多数没有忠实地重述MPM肿瘤微环境15，16，17，18的复杂方面。在小鼠、基因工程MPM小鼠模型或鼠MPM细胞系协同移植模型中使用石棉诱导的MPM受到基本表型和功能差异的限制，因此，将新发现转化为临床效果不佳。其他临床前小鼠MPM模型主要依靠免疫缺陷小鼠的人类细胞系的皮下或围肠异种移植物。虽然这些模型很容易监测和提供基本数据，但这些异种移植物的微观环境并不与人类肿瘤相媲美，损害大多数这些临床前研究的转化能力17，19。相反，正交异种移植更好地反映患者的肿瘤行为和治疗反应，因为他们被一个类似的微环境包围，在原始肿瘤部位16发现。
分子成像由+18F_FDG-PET/CT是纵向监测MPM20，21患者疾病进展的首选方法。因此，采用这种非侵入性成像方法，极大地促进了临床前研究转化为临床试验16，22。此外，它有助于减少所需的动物数量，因为每只动物代表自己的控制随着时间的推移。
在本文中，我们提出了一个可靠的正交异种异种移植MPM模型后，获得的人MPM细胞系H2052/484注射到胸腔的贫血小鼠。结合18F_FDG-PET/CT成像，该模型是研究新的诊断策略和治疗人体MPM的功能和机械效应的宝贵和可重复的方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3-mice bed</td>
			<td>Minerve</td>
			<td></td>
			<td>bed for mice imaging</td>
		</tr>
		<tr>
			<td>Athymic Nude-Foxn1n nu/nu</td>
			<td>Envigo, Huntingdon, UK</td>
			<td>6907F</td>
			<td>immunodeficient mouse</td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Mundipharma Medical Company, CH</td>
			<td>111131</td>
			<td>polyvidone iodine solution</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline (DPBS)</td>
			<td>ThermoFisher Scientific, Waltham, MA, USA</td>
			<td>14190094</td>
			<td>Buffer for cell culture</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>PAA Laboratories, Pasching, Austria</td>
			<td>A15-101</td>
			<td>cell culture medium supplement</td>
		</tr>
		<tr>
			<td>Insulin syringes</td>
			<td>BD Biosciences, San Jose, CA, USA</td>
			<td>324826</td>
			<td>syringe for cell injection</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>ThermoFisher Scientific, Waltham, MA, USA</td>
			<td>15140122</td>
			<td>antibiotics for cell culture medium</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>ThermoFisher Scientific, Waltham, MA, USA</td>
			<td>61870010</td>
			<td>basal cell culture medium</td>
		</tr>
		<tr>
			<td>Temgesic (Buprenorphin 0.3 mg/mL)</td>
			<td>Alloga SA, CH</td>
			<td>700320</td>
			<td>opioid analgesic product</td>
		</tr>
		<tr>
			<td>Triumph PET/SPECT/CT</td>
			<td>Trifoil, Chatsworth, CA, USA</td>
			<td></td>
			<td>imaging equipment</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>ThermoFisher Scientific, Waltham, MA, USA</td>
			<td>25050014</td>
			<td>enzymatic cell dissociation buffer</td>
		</tr>
		<tr>
			<td>Virkon S 2%</td>
			<td>Milian, Vernier, CH</td>
			<td>972472</td>
			<td>disinfectant</td>
		</tr>
		<tr>
			<td>Vivoquant</td>
			<td>Invicro, Boston, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

implantation and monitoring by pet/ct of an orthotopic model of human pleural mesothelioma in athymic mice

恶性胸膜内皮瘤（MPM）是一种罕见的、具有攻击性的肿瘤，在覆盖肺部、心脏和胸腔的中皮内出现。MPM 开发主要与石棉相关。治疗只能提供适量的存活率，因为中位平均存活率从诊断时起为9-18个月。因此，必须确定更有效的治疗方法。描述新治疗靶点的大多数数据都是从体外实验中获得的，需要在可靠的体内临床前模型中进行验证。本文介绍了一种在将人类MPM细胞系H2052/484注射到免疫缺陷贫血小鼠胸腔后获得的这种可靠的MPM正位模型。在正交位位移植允许研究肿瘤在自然体内环境的进展。正电子发射断层扫描/计算机断层扫描（PET/CT）分子成像使用临床[18F+-2-氟-2-脱氧-D-葡萄糖（=18F_FDG）放射跟踪器是检查MPM患者的首选诊断方法。因此，使用18F_FDG-PET/CT 纵向监测 H2052/484 正射模型的疾病进展。这种技术具有很高的3R潜力（Reuce动物的数量，Refine以减轻疼痛和不适，和Replace动物实验与替代品），因为肿瘤的发展可以监测非侵入性和动物的数量可以大大减少。
该模型显示高开发率，快速肿瘤生长，是成本效益，并允许快速临床翻译。通过使用这个正交异种性异种性MPM模型，研究人员可以评估治疗干预后可靠MPM模型的生物反应。

H2052/484 正射模型 通过胸腔内注射培养癌细胞，特别是H2052/484细胞进行正交MPM模型，设置相对容易。上述不同步骤只需要适度的细胞培养知识，而中等训练的动物实验者可以进入手术步骤。裸体小鼠和细胞应在无菌条件下操作，以最大化植入的结果。通过仔细遵循这个方案，包括短暂的麻醉和最小的手术，我们只遇到1死亡在266小鼠注射不同的MPM细胞系。在266只被仔细注射的小鼠中?...

Watch this Scientific Journal Video about Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice at JoVE.com

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice

本文介绍了通过将H2052/484中皮瘤细胞植入免疫功能性贫血小鼠胸腔，生成人类胸膜中皮瘤的正交小鼠模型。通过非侵入性多式联运 =18F_-2-fluo-2-脱氧-D-葡萄糖正电子发射断层扫描和计算机断层成像，对胸腔内肿瘤的发展进行了纵向监测。

小鼠中人类胸膜皮质瘤的PET/CT的植入与监测

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. ...

该方法有助于回答与人类胸膜间皮瘤的发育、治疗和诊断有关的关键问题。这种可靠的临床前正畸模型的主要优点是，它复制人类疾病进展和病理学在微环境接近一个发现在胸膜间皮瘤患者。因此，这一无价的模型具有很高的意义，使用非侵入性分子成像可以按照三个R概念进行纵向监测。在开始准备麻醉系统和手术区之前，通过喷洒消毒剂的所有表面，在层流罩中。将微隔离的 SPF 保持架放在消毒流罩中。将加热垫、波维酮碘溶液 30 量汉密尔顿注射器、纱布和棉签手术器械以及微管和尖端放在层流罩中。一旦小鼠被正确麻醉皮下注射0.05毫克每公斤布普里诺芬为术后锅缓解。然后将鼠标放在加热垫顶部的右侧，用波维酮碘溶液清洁手术区域，并制作5毫米的皮肤切口。用钝剪刀清除周围的脂肪和肌肉，露出肋骨。使细胞悬浮液均匀化，将50微升装入汉密尔顿注射器，确保避免气泡。每次注射前用 70% 酒精擦拭针头。慢慢地将细胞注射到第六和第七根肋骨之间的胸腔中，在骨质肌肉下以30度和2到3毫米的角度拿着针头。将针头留在肋骨下，避免注射到肺部。它应该通过肌肉可见。完成后，用三到四个可吸收的缝合线关闭伤口，将鼠标存放在温暖的环境中，直到它醒来。进行植入时，正确定位针头以限制针头穿透深度非常重要。在注射氟-18（FDG）之前，使用加热室加热垫或红外灯在30摄氏度下预热小鼠，使用剂量计算器在1毫升胰岛素注射器中准备3至4个巨型贝克雷尔剂量的氟-18（FDG）。。用150至200微升盐水注射。确保记录所有时间放射性剂量测量注射，和PET扫描，以计算标准吸收值或SUV。称重小鼠，然后静脉注射氟-18（FDG）后注射让小鼠在温暖的条件下清醒45分钟。然后，将鼠标加载到扫描仪床上，将床转移到扫描仪上，并让动物接受以肺部为中心的CT扫描。将床移动到 PET 子系统，在氟-18（FDG）喷射一小时后插入采集，持续时间为 15 分钟。然后将小鼠从成像室中取出，并允许它们在笼子里恢复，将它们放在一个专门用于放射性衰变的区域。在图像分析之前，重建CT和PET扫描，如手稿中所述。通过扫描幻象圆柱来校准图像，并根据内置的软件解决方案自动注册扫描。通过单击打开的数据图标来分析图像加载 CT 数据作为参考。然后通过单击追加数据图标加载 PET 数据作为输入。调整色度，使 CT 和 PET 对图像进行对比，以便进行目视检查。从下拉菜单中选择 3D ROI 工具，单击"添加 ROI"，并命名文件肺。单击分段算法和邻域阈值，然后将输入定义为背景，将图像定义为 ref。根据小鼠肺密度值输入最小值和最大值。单击 VTK 图标检查 3D 渲染的肺。然后单击"显示表"图标并检索生成的表中的卷。要分析肿瘤中的氟-18（FDG），请从下拉菜单中选择荧光粉，将PET图像转换为SUV。选择标量乘数，然后使用 NP 1 作为选定，并设置每毫升贝克雷尔到 SUV 因子作为标量。最后从下拉菜单中选择 3D ROI 工具，然后单击添加 ROI 并命名文件肿瘤。单击 3D 绘画模式和球体仅取消选中 2D，并调整形状的大小以包围肿瘤。单击显示表图标并从生成的表中检索 SUV 最大值。CT 扫描的 3D 渲染概述了 MPM 肿瘤定位，并允许计算肺体积。注射宫内肿瘤后，肺体积测量会随着时间的推移显著减少。PET扫描提供了有关MPM肿瘤代谢状态的宝贵信息。移植后两周肿瘤是可分辨的，通过提取与注射后天数呈正相关数的SUV来量化氟-18（FDG）吸收。此外，肺体积和氟-18（FDG）的特异性与0.6的R平方相互关联，支持这些测量的强度，以监测MPM正畸肿瘤的发展。按照这个程序，其他方法，如组织学，免疫组织化学，流细胞学可以执行，以响应正畸问题，如肿瘤和微环境的增殖状态和表型特征。总之，这些临床前技术为研究人员探索胸膜间皮瘤的诊断和治疗新策略铺平了道路。此外，分子成像的使用需要迅速将新发现翻译到诊所。

Research

JoVE Journal

Immunology and Infection

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

浆液性卵巢癌原位模型在免疫小鼠，为在体内肿瘤显像和肿瘤免疫反应的监测

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

科研

免疫与感染

Comprehensive &amp; Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

Comprehensive & Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

艾滋病毒/艾滋病的综合成本有效的实验室监测：非洲的角色模型

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

移植物抗宿主病的诱导和<em在体内</em&gt; T细胞监测中的应用MHC匹配的小鼠模型

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK

复发性单纯疱疹性角膜基质性角膜炎小鼠模型研究人类HSK

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical ...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

一个<em在体外</em&gt;模型研究人类巨噬细胞分化和极化的异质性

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

Optimized Protocols for Mycobacterium leprae Strain Management: Frozen Stock Preservation and Maintenance in Athymic Nude Mice

Optimized Protocols for Mycobacterium leprae Strain Management: Frozen Stock Preservation and Maintenance in Athymic Nude Mice

优化的协议<em&gt;麻风分枝杆菌</em&gt;应变管理：冷冻库存保持和维护在无胸腺裸鼠

Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

半自动化的方法准备抗体鸡尾酒人外周血中免疫表型分析

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

小鼠淋巴细胞通过标签<sup&gt; 64</sup&gt;的Cu-抗体受体靶向用于<em&gt;体内</em&gt;由PET / CT细胞运输

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

使用的淋巴细胞外渗分析<em&gt;体外</em&gt;人类血脑屏障模型

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

IL-8瞬时转化的小鼠模型<em&gt;体内</em&gt;长期监测炎症反应

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...