这项工作是由格兰特 - 在援助从日本学术振兴会KAKENHI为AEH（批准号25461196）和TB（授权号码23390218和15H04833）和国家卫生研究院资助HL111190的（DMO）的支持。作者要感谢山本美雪为她与动物基因分型和组织切片的准备帮助努力。我们感谢合作研究资源，医学院，庆应义塾大学为技术依托和试剂。

动物实验是由庆应义塾大学的机构动物护理和使用委员会批准。 注:在本研究中，我们使用了SFTPC-rtTA和TRE-Fgf9基因-IRES-EGFP DT小鼠中，肺腺癌迅速被喂食含有强力霉素6,7州城诱导后发展起来的。然而，所有的评估程序可以应用于其他肺癌小鼠模型。 1.实验大纲: <ol><li>确定在基线肺的状态: <ol><li>肿瘤诱导前，当DT老鼠是8 - 19周龄，执行第一台微型CT扫描（见下文第2和3）。这可作为肺基线扫描，确认没有造成漏水转基因自发研制的结节，并记录肿瘤诱导前没有任何现有的肺部病理。 </li></ol></li><li>启动肿瘤的诱导: <ol><li>开关与常规CH的DT鼠标流以强力霉素饲料以诱导成纤维细胞生长因子（FGF）9的表达在肺泡细胞并以引发肿瘤的发展。给周星驰强力霉素（200ppm的）自由采食。 </li></ol></li><li>确认肿瘤结节的小鼠肺中的发展: <ol><li>执行微CT扫描，以确定相对于感应预扫描肿瘤结节的发展。 </li></ol></li><li>评估对治疗的反应: <ol><li>为了测试显微CT的检测肿瘤结节的变化对治疗的反应，施用FGF受体（FGFR）抑制剂AZD4547的能力，那么5和10周后进行额外的微CT扫描。 </li></ol></li><li>终点的评价: <ol><li>安乐死的组织学评估（见下文第6节）的所有治疗和控制小鼠和过程的组织。 </li></ol></li></ol> 2.显微CT图像采集准备小鼠: <ol><li>打开微CT扫描仪和计算机。 </li><li> CLIC钾对名为"R_m CT2"的软件，然后点击"热身"。 </li><li>取出样品床在其上鼠标将从显微CT室被放置。裹在床上用保鲜膜。 </li><li>通过添加异氟醚麻醉蒸发器到显着水平设置麻醉诱导框。设定在3升/分钟的异氟醚的流速。 </li><li>打开氧气罐以启动氧气流入感应箱，并设置在1升/分钟的流速。 </li><li>放置在麻醉诱导框鼠标，并确认它是深受没有响应于皮肤捏自发运动的和（皮肤的小褶皱，这不会导致组织损伤或皮肤破的温和捏）麻醉。 </li><li>应用眼部润滑剂，以防止在麻醉过程中角膜干燥。 </li><li>打开微CT室，并将与背侧鼠标向上的样品床。按住鼠标的头部，并从在奥德下肢向下拉动体R以伸展和对称地挺直身体。 </li><li>关掉麻醉流动到感应箱并打开向连接到显微CT室中的管。 </li><li>将麻醉管插入老鼠的鼻子管理连续麻醉剂。 </li><li>为了解决鼠标的位置;包鼠标和用保鲜膜样品床。 注:关键是要保持鼠标下深麻醉并包裹到样品床，因为在扫描过程中任何轻微的运动或它的身体扭会导致朦胧的图像和解释的难度。 </li><li>关闭微CT室。 </li><li>调整微CT系统90千伏和160微安和扫描时间4.5分钟。设置图像范围24×19毫米和体素大小为50×50×50微米。使用了心跳同步模式。 </li><li>使"数据库"的新文件夹，以便该文件夹中保存新的图像。 </li><li>开始扫描。 </li><li>扫描完成后，将鼠标移动到一个空笼子，直到它恢复意识观察它。不要把它与其他老鼠，直到它已完全麻醉的影响中恢复。 </li></ol> 3.预感应显微CT图像可视化和分析: <ol><li> :以可视化的显微CT图像，从以下网站下载免费软件的ImageJ <a href="http://imagej.nih.gov/ij/">http://imagej.nih.gov/ij/</a> 。注意:每个显微CT数据文件大约为500 TIF文件的堆栈（.TIF）。其它图像浏览软件也可使用。 </li><li>打开串行显微CT图像文件，并通过所有各小鼠的从颈部图像的滚动到腹部或反之亦然。 </li><li>使用幼稚野生型小鼠的扫描，识别不同的区域和正常的解剖结构的密度在基于鼠标解剖学知识胸部（ 见图1A）。 <ol><li>将光标与计算机鼠标和滚动，从白色腹腔脏器和隔膜，通过胸部和高达颈部开始。 </li><li>确定骨胸廓地标（胸骨在前面，脊椎的背部和肋骨两侧）。 </li><li>确定在胸部的前面和主要血管邻近心脏和在纵隔心脏。 </li><li>观察（在颈部和上胸部的水平作为小暗圈），其分叉成右和左主支气管然后继续分支成越来越小支气管气管内腔。请注意，每个支气管密切有两个或三个血管（ 图1A）相关联。 </li></ol></li><li>启动检查未诱导DT小鼠的扫描，并确定任何异常是否存在。 <ol><li>排除与任何进一步的实验异常预诱导肺部阴影（如结节，肺大疱肺气肿等 ）的小鼠。 （ 图1C-E，G，H </s仲&gt;）。 </li></ol></li></ol> 4.肿瘤诱导: <ol><li>为了诱导实验小鼠肿瘤的发展是显示正常肺扫描，切换他们的食物从常规食物含多西环素州城（200ppm的）。 </li></ol> 5.后续扫描: <ol><li> 10周后，执行所有小鼠的第二个微CT扫描，以确认肿瘤结节在其肺（参见图2）的发展。 </li><li>分裂的小鼠分成两组。 （通过胃管6天/周10周125微克/ kg /天）到一组和安慰剂到另一对照组为10个星期管理该FGFR受体阻滞剂AZD4547。 </li><li> 5周后 - 4进行第三次扫描跟进的结节状阴影的变化。 </li><li> 10周的治疗结束时，执行第四扫描。 </li><li>安乐死用CO 2吸入或以0.1毫克/ 200微升戊巴比妥腹膜内注射所有小鼠。 </li><li>至确定在诱导后，并且响应于治疗的肿瘤结节的动态变化，在两个或更多个不同的时间点标识相同小鼠的串行扫描图像内的类似位置，然后检查外观/任何异常阴影消失（ 图3 ）。 </li><li>为了便于在同一平​​面的在不同时间点相同的鼠标的识别，尽量感兴趣的平面与小鼠胸内解剖标志的结构相关联。 <ol><li>使用地标，如气管，分叉，左，右主支气管，主动脉，隔膜和大血管。 注:胸部的骨骼，包括胸椎，肋骨，胸骨和作为是因为在鼠标主体对准一般轻微倾斜的样品床，增加了扫描位置的误解的可能性，定位地标不太有用。同样地，在扫描文件内的图像的序列号是不可靠identifyin克相同位置随着时间的推移，因为在小鼠体长从一个时间点到另一个的改变。故障或不准确识别在不同时间点相同的平面，可能会导致发现的假阳性/阴性的解释。 </li></ol></li></ol> 6.鼠标安乐死和肺收藏: <ol><li>安乐死的小鼠用CO 2吸入或以0.1毫克/ 200微升戊巴比妥腹膜内注射。 </li><li>通过腹壁纵向切割露出腹腔脏器。流血鼠标通过解剖腹主动脉以降低血液中的肺容积。 </li><li>缝细剪的隔膜;这将导致从胸腔的负压的损失，从而塌陷肺部。通过切割和去除前胸壁的肋的部分露出肺和心脏。通过切割皮肤和软组织以暴露trache清洁颈部的前部一个。 </li><li>切开心脏和胸腺。插入气管后面镊子将其从食道分开。 </li><li>导管插入用G24插管气管然后通过拧紧围绕着插入部的螺纹固定到位。 </li><li>膨胀并用25厘米柱通过气管套管用冰冷的4％多聚甲醛（PFA）固定肺。分离套管，并拧紧螺纹，以防止煤灰泄漏然后被切断的上气管关闭其附着于喉。 </li><li>从缝合线拉气管和从其附着解剖它，向下继续与肺烯 -嵌段移除。将其插入到含有5ml 4％PFA的15毫升管。留在PFA O / N肺部，以确保完成组织渗透和固定，然后处理成组织标准的石蜡块8。 </li><li>切蜡块到6微米厚的切片在切片和染色与HE染色使用标准技术。 </li></OL&gt; 7.组织学评价: 注意:虽然在此描述了使用一种"幻灯片扫描器"用于数字组织学评价的，用于评估的使用经常显微镜和视觉组织学评价也是可能的。 <ol><li>打开滑动扫描器仪器和计算机。 </li><li>点击"扫描NDP"软件。 </li><li>选择扫描模式"幻灯片批"和"半自动模式"扫描一系列幻灯片。 注意:连续扫描期间拿起幻灯片机械臂是在玻璃载片的边缘的任何不规则非常敏感。 </li><li>要将其装入机器之前触诊所有载玻片的边缘。如果有盖玻璃的任何突起或干燥安装介质，用切割或手术刀清洁它关闭。 </li><li>幻灯片加载到幻灯片卡带。打开标本舱口盖，纸盒滑入到位"一";。关上门。 </li><li>在计算机软件，给予简短的描述性名称的所有幻灯片在磁带的相应位置，然后单击"确定"按钮。 </li><li>选择配置模式:"明场"。 </li><li>点击"开始批"开始扫描暂定。 注意:一旦机器完成扫描所有的幻灯片，软件会自动检测到的所有幻灯片与组织区域，并建议把它作为关注区域。 </li><li>如果需要，通过按住鼠标左键并拉动区域边界重新定义感兴趣的区域。 注:定义过大面积的幻灯片作为感兴趣的区域将导致扫描更长的时间。 </li><li>一旦满意利益上的所有幻灯片的区域，点击"扫描"开始扫描所有幻灯片。 注:扫描文件可以数字方式低分辨率和高分辨率观察和图像可以导出为JPEG文件。 </li></ol>

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The authors declare that they have no competing financial interests.

此处描述了用于肺部异常的实时识别和监测肿瘤结节的发展和在肺癌动物模型中对治疗的反应将使谁正在进行肺癌相关实验计划更多的科学家的基于显微CT-方法准确，高效的实验，同时节省时间和资源。我们以前使用的MRI为同一目的6。扫描和阈值检测肺结节的MRI的清晰度均低于那些在这项研究中6中所述的微型CT扫描。 即使用了类似的肺腺癌小鼠模型（具有不同的遗传背景和肿瘤诱导方法）以前的研究显然有这样的本来是可以避免或改善他们使用了显微CT扫描9,10几个缺点。他们没有办法确定任何预诱导肺部异常或结节，从而冒着混淆他们的结果，列入不适合动物。他们也没有办法来确认肿瘤诱导后或传播癌细胞进入野生型小鼠（以诱导的继发性肿瘤）之后的发展，使他们不得不等待4-8个月，然后盲目地安乐死小鼠的组织学鉴定肿瘤。当这些模型被用来评估药物的治疗潜力，研究人员不得不分配若干组在一个时间顺序被安乐死以检测对肿瘤结节的治疗效果与组织学和能够识别的最大效应的时间点11。 我们的方法的一个限制，但是，是掩盖在肺部结节的检测当一些外来物质被注入气管7（未示出数据）朦胧阴影的外观。一些小鼠似乎发展到注射物质延长炎症反应，这reacti的阴影并伴随间质水肿掩盖现有的结节。在这种情况下，MRI可以尝试作为替代。 最近，其他成像方式相继出台，以评估小动物各种病症;像生物发光成像12和正电子发射断层扫描（PET）13。据我们所知，这些成像系统还没有被用于尚未评估在小鼠模型肺肿瘤如此的检测效率和图像的清晰度与小动物MRI和微CT图像比较尚不可能。这些机器的可用性和成本是它们的广泛使用的限制因素。 我们检测肺部异常小鼠的约50％在感应预检并相应地这些小鼠被排除进一步的实验。这种高的异常发生率可能会在一些动物中"渗漏"FGF9转基因导致肿瘤结节的发展的结果就是他们有致，甚至14之前。事实上，当组织学检查大约一半的排除动物表现出的多个小结节的存在。这些也可以通过插入的转基因在小鼠基因组14中的邻近基因的异常效应引起的。这些排序异常的发展绝不特定于SFTPC-rtTA和TRE-Fgf9基因-IRES-EGFP的双转基因小鼠，因此其他肺癌模型可能从类似的情况是患。这种高异常亮点发生偶数更多采用显微CT小鼠纳入预检的重要性。天真野生型小鼠从未表现出对显微CT（图1A）任何不正常的阴影。 总之，我们已经描述了一种利用一个小动物显微CT机，用于肿瘤结节中的腺癌小鼠模型的肺部发育的精确检测和监测的方法。通过用它来监视随时间的结节的变化，更精确的数据可以被收集，并具有成本和时间有效的时间当然可以适应。

肺癌是癌症死亡的世界1的周围的首要原因。研究预防，早期发现和肺癌的治疗是在世界各地的许多2,3研究中心正在进行。几种动物模型为肺癌已经开发，并且它们已被证明在研究肺致癌作用和来源的细胞的机制，在确定癌干细胞的存在是有用的，并且在研究各种新的治疗策略4。早期型号小鼠5的敏感菌株依靠致癌物诱发肿瘤发生。敲除和转基因小鼠模型，其中肺癌产生所具体操纵遗传病变的结果的发展已经大大提高了我们对控制肿瘤诱导和模拟几种人类肺癌4的各方面的能力。然而，在使用肺癌动物模型的一个主要挑战是在不存在实时的方法来准确地识别和监测肿瘤的小鼠肺的发生和发展，并记录下它们的大小，以后的任何改变，如它们在治疗反应持续增长或减少。这就迫使研究诉诸几个时间，精力和资源消耗的技术，以确定肿瘤，并评价它们的实验结果。固有小鼠间变化的响应于肿瘤诱导的存在需要在每个实验组使用大量动物的减少数据的变异性。无法评估实时的肿瘤生长或对治疗的反应，迫使研究者盲目安乐死在延长的实验方案的多个时间点的小鼠，以保证它们将收集的正确的数据，造成资源的从样品中的废物在该要么过早或过晚的时间点收集。 在目前的研究中，一种方法利用小动物微-Computed断层（微CT）扫描仪来检测和后续肺肿瘤活小鼠被引入。我们用我们最近描述SFTPC-rtTA和TRE-Fgf9基因-IRES-EGFP双转基因（DT）小鼠迅速发展肺腺癌诱导后用强力霉素6,7。使用显微CT的，使我们能够（除其他外）排除与异常肺部异常小鼠诱导前，确认在肺肿瘤结节的发展诱导后，并且响应于试验性治疗观察肿瘤结节的变化。小鼠和组织学评估的终点安乐死证实显微CT进行的实时评估的准确性。我们相信，这一技术铺平了道路实施使用肺癌动物模型，同时节省了宝贵的资源，缩短观察时间，提高结果的准确性和了解规划更好的实验。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">micro-X-ray&#8211;computed tomography</td>
			<td style="width:264px;">Rigaku</td>
			<td style="width:140px;">R_mCT2</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoZoomer RS Digital Pathology System</td>
			<td style="width:264px;">Hamamatsu&#160;</td>
			<td>RS C10730</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NDP.view2&#160;Viewing software</td>
			<td style="width:264px;">Hamamatsu&#160;</td>
			<td>U12388-01</td>
			<td>http://www.hamamatsu.com/jp/en/U12388-01.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Isoflurane Vaporizer - Funnel-Fill</td>
			<td style="width:264px;">VETEQUIP</td>
			<td>911103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Induction chamber, 2 Liter&#160; W9.5&#215;D23&#215;H9.5</td>
			<td style="width:264px;">VETEQUIP</td>
			<td style="width:140px;">941444</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Isoflurane</td>
			<td style="width:264px;">Mylan</td>
			<td>ES2303-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AZD 4547</td>
			<td>LC Labratories</td>
			<td>A-1088</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pentobarbital</td>
			<td style="width:264px;">Kyoritsu</td>
			<td style="width:140px;">SOM02-YA1312</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">G24 cannula&#160;</td>
			<td style="width:264px;">Terumo</td>
			<td style="width:140px;">SP-FS2419</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td style="width:264px;">Wako</td>
			<td style="width:140px;">163-20145</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Microtome</td>
			<td style="width:264px;">Leica</td>
			<td style="width:140px;">RM2265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Doxycycline</td>
			<td style="width:264px;">SLC Japan/PMI Nutrition International</td>
			<td style="width:140px;">5TP7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageJ software&#160;</td>
			<td style="width:264px;">National Institute of health</td>
			<td style="width:140px;"></td>
			<td>http://imagej.nih.gov/ij/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Puralube vet ointment (Occular lubricant)</td>
			<td style="width:264px;">Dechra</td>
			<td style="width:140px;">NDC 17033-211-38</td>
			<td></td>
		</tr>
	</tbody>
</table>

using micro-computed tomography for the assessment of tumor development and follow-up of response to treatment in a mouse model of lung cancer

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse models of lung cancer are being used to study the mechanism of cancer development and to experiment with various therapeutic strategies. However, the absence of a real-time technique to identify the development of tumor nodules in mice lungs and to monitor the changes in their size in response to various experimental and therapeutic interventions hampers the ability to obtain an accurate description of the course of the disease and its timely response to treatments. In this study, a method using a micro-computed tomography (CT) scanner for the detection of the development of lung tumors in a mouse model of lung adenocarcinoma is described. Next, we show that monthly follow-up with micro-CT can identify dynamic changes in the lung tumor, such as the appearance of additional nodules, increase in the size of previously detected nodules, and decrease in the size or complete resolution of nodules in response to treatment. Finally, the accuracy of this real-time assessment method was confirmed with end-point histological quantification. This technique paves the way for planning and conducting more complex experiments on lung cancer animal models, and it enables us to better understand the mechanisms of carcinogenesis and the effects of different treatment modalities while saving time and resources.

在基线进行与肺部异常的小鼠的鉴定。肿瘤诱导，当DT小鼠8前 - 12周龄，所有小鼠的肺用显微CT扫描。令人惊讶地，小鼠的约50％的显示，迫使我们认为它们不适合纳入在随后的研究异常。这些异常是结节状阴影，大单或多个小肺气肿肺大疱和/或肺不张肺叶（ 图1A，C，DE，GH）。然后，FGF9转基因和肿瘤的诱导作用activated.Only小鼠表现出正常的肺扫描（无异常）的常规食物中切换到州城强力霉素诱导肺泡细胞和随后肿瘤发展FGF9的表达。以确认肿瘤结节在小鼠肺10周后从肿瘤诱导开始发展，后续微CT扫描进行。这些发现为D在所有小鼠可变尺寸的多个结节才有发展（ 图2，比较A到B和D至E）。进行微CT扫描，以评估对治疗的反应。这些表明，该施用的FGFR抑制剂AZD4547 10周确实表现出在分别在预处理扫描（ 图 3可见结节的尺寸减少或消失，将小鼠比较前，诱导后和后处理， 交 ，EG和IK）。另一方面，那些没有给予FGFR抑制剂和给予安慰剂的对照小鼠，而不是表明没有变化，增加结节尺寸和新的结节外观（ 图3，MO）;确认在尺寸减小的治疗组中观察到的是治疗干预的一个真正的效果。 图1B，F，I）。在所有动物（ 图2C，F）观察到具有可变大小和位置的多个结节腺癌:12周诱导多西环素- 10后的小鼠。小鼠经过10 - 12周强力霉素诱导的随后用10 - 19星期内FGFR阻滞剂:结节在数量少得多和更小（ 图3D，H，L）相比，这些观察到的诱导后。 12周强力霉素诱导后10 - - 10后小鼠12周安慰剂:多个大结节可见（ 图3P）<html>类似于这些在诱导后时间点看到。 <img alt="图1" src="/files/ftp_upload/53904/53904fig1.jpg" /> 图1显微CT标识预诱导肺部异常，所有小鼠用显微CT强力霉素诱导开始之前检查。当检测到肺部异常，小鼠安乐死用于组织学确认。为（A）一种正常肺代表性显微CT扫描图像;（C）与萎陷区的肺;（DE）与肺气肿大疱和具有异常结节阴影（GH）肺的肺。组织学评估确认微型CT检查结果的准确性:（B）正常肺组织;（六）多肺气肿区域;和我）的多个肿瘤结节。比例尺:200μm左右。53904fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/53904/53904fig2.jpg" /> 图2.显微CT标识诱导。小鼠后肿瘤结节即显示出清洁预感应扫描饲喂强力霉素饲料10周，然后用显微CT重新评估，之后将其安乐死用于组织学评估的发展 。从两个不同的小鼠的代表性显微CT扫描图像显示多个结节状阴影强力霉素州城开始10周后的外观。（A，D）预感应干净的肺，（B，E）相同位置扫描图像显示的发展多发结节（红色箭头）。小黄色箭头指向用于识别在不同的时间点相同的位置的解剖标志。（C，F）伴有结节看出在微CT扫描为Confirmed在肺组织切片。比例尺:200微米<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53904/53904fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53904/53904fig3.jpg" /> 图3.显微CT标识更改在肿瘤结节中对治疗的反应。的小鼠显示出清洁感应预扫描饲喂强力霉素饲料10周，然后进行一个后感应显微CT。接着，它们被施用的FGFR抑制剂AZD4547 10个星期，用显微CT（治疗后）重新评估。对照小鼠给予安慰剂代替的FGFR抑制剂澄清治疗干预的实际效果。最后，将小鼠安乐死用于组织学评价。 代表性显微CT扫描图片来自四个不同的小鼠（A，E，I，M） </str翁&gt;预感应扫描证实小鼠均无异常（B，F，J，N）从同一小鼠扫描它们用强力霉素饲料10周诱导后。所有呈多结节（红色箭头，红色的椭圆形包围之前由结节已经完全被遮挡的区域）。（C，G，K）额外10周的FGFR抑制剂治疗之后，从相同的小鼠扫描显示完全消失或显着减少在结节的尺寸。（O）从对照小鼠扫描到表示施用安慰剂代替FGFR抑制剂表示肿瘤结节&#39;的大小和数量增大，对照组。小黄色箭头指向用于识别在不同的时间点相同的位置上的解剖学标志（D，H，L）组织切片确认在结节的大小和数量的显着减少（比较图2 C和F）。（P） 的显示prese多种肿瘤结节NCE当安慰剂管理。比例尺:200微米<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53904/53904fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer at JoVE.com

Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer

采用微计算机断层扫描对肿瘤发展的评估和后续反应来治疗肺癌的小鼠模型

We describe a method for the detection of tumor nodule development in the lungs of an adenocarcinoma mouse model using micro-computed tomography and its use for monitoring changes in nodule size over time and in response to treatment. The accuracy of the assessment was confirmed with end-point histological quantification.

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse ...

using-micro-computed-tomography-for-assessment-tumor-development

Nanyang Technological University

Research

JoVE Journal

Medicine

10.6K 次观看.  Keio University School of Medicine. We describe a method for the detection of tumor nodule development in the lungs of an adenocarcinoma mouse model using micro-computed tomography and its use for monitoring changes in nodule size over time and in response to treatment. The accuracy of the assessment was confirmed with end-point histological quantification.

视频: Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.

新型抗癌药物使用手术标本肿瘤植的发展方法


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

科研

医学

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for imaging vasculature, few are able to visualize the vascular network as a whole while extending to a resolution that includes the smaller vessels1,2. Additionally, many vascular casting techniques destroy the surrounding tissue, preventing further analysis of the sample3-5. One method which circumvents these issues is micro-Computed Tomography (&mu;CT). &mu;CT imaging can scan at resolutions &lt;10 microns, is capable of producing 3D reconstructions of the vascular network, and leaves the tissue intact for subsequent analysis (e.g., histology and morphometry)6-11. However, imaging vessels by ex vivo &mu;CT methods requires that the vessels be filled with a radiopaque compound. As such, the accurate representation of vasculature produced by &mu;CT imaging is contingent upon reliable and complete filling of the vessels. In this protocol, we describe a technique for filling mouse coronary vessels in preparation for &mu;CT imaging.
Two predominate techniques exist for filling the coronary vasculature: in vivo via cannulation and retrograde perfusion of the aorta (or a branch off the aortic arch) 12-14, or ex vivo via a Langendorff perfusion system 15-17. Here we describe an in vivo aortic cannulation method which has been specifically designed to ensure filling of all vessels. We use a low viscosity radiopaque compound called Microfil which can perfuse through the smallest vessels to fill all the capillaries, as well as both the arterial and venous sides of the vascular network. Vessels are perfused with buffer using a pressurized perfusion system, and then filled with Microfil. To ensure that Microfil fills the small higher resistance vessels, we ligate the large branches emanating from the aorta, which diverts the Microfil into the coronaries. Once filling is complete, to prevent the elastic nature of cardiac tissue from squeezing Microfil out of some vessels, we ligate accessible major vascular exit points immediately after filling. Therefore, our technique is optimized for complete filling and maximum retention of the filling agent, enabling visualization of the complete coronary vascular network &#x2013; arteries, capillaries, and veins alike.

Visualization of the coronary vessels is critical to advancing our understanding of cardiovascular diseases. Here we describe a method for perfusing murine coronary vasculature with a radiopaque silicone rubber (Microfil), in preparation for micro-Computed Tomography (&mu;CT) imaging.

微型计算机断层扫描成像制备小鼠冠状动脉血管逆行灌注和灌装

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for ...

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

测量和制止在弱视治疗

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

原位膀胱肿瘤模型和评价治疗膀胱萨纳

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

对黑色素瘤修饰符一个斑马鱼原地肿瘤模型的筛选

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

一种新型的高分辨率<em&gt;在体内</em&gt;成像技术研究肿瘤生长及治疗颅内结构的动态响应

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

药物输送到微型计算机断层扫描成像缺血后肢血管灌注与微滤渗透

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Established cancer cell lines and xenografts have been the mainstay of cancer research for the past several decades. However, recent evidence suggests that therapeutic response is greatly influenced by the tumor cell microenvironment. Therefore, we have developed an ex vivo analysis of primary tumor specimens for drug development purposes.

原发性肿瘤和/或转移相关的临床前和治疗的临床开发的离体处理响应

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

评估人体脂肪注射生存能力的裸鼠与微型计算机断层扫描

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...