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>

<ol>
	<li>Ferlay, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+incidence+and+mortality+worldwide:+sources,+methods+and+major+patterns+in+GLOBOCAN+2012.">Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012.</a> Int. J. Cancer. 136, 359-386 (2015).</li><li>Mak, I. W., Evaniew, N., Ghert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lost+in+translation:+animal+models+and+clinical+trials+in+cancer+treatment.">Lost in translation: animal models and clinical trials in cancer treatment.</a> Am. J. Transl. Res. 15, 114-118 (2014).</li><li>Chen, Z., Fillmore, C. M., Hammerman, P. S., Kim, C. F., Wong, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-small-cell+lung+cancers:+a+heterogeneous+set+of+diseases.">Non-small-cell lung cancers: a heterogeneous set of diseases.</a> Nat. Rev. Cancer. 14, 535-546 (2014).</li><li>Kwon, M. C., Berns, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+for+lung+cancer.">Mouse models for lung cancer.</a> Mol. Oncol. 7, 165-177 (2013).</li><li>Malkinson, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetic+basis+of+susceptibility+to+lung+tumors+in+mice.">The genetic basis of susceptibility to lung tumors in mice.</a> Toxicology. 54, 241-271 (1989).</li><li>Yin, Y., Betsuyaku, T., Garbow, J. R., Miao, J., Govindan, R., Ornitz, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+induction+of+lung+adenocarcinoma+by+fibroblast+growth+factor+9+signaling+through+FGF+receptor+3.">Rapid induction of lung adenocarcinoma by fibroblast growth factor 9 signaling through FGF receptor 3.</a> Cancer Res. 73, 5730-5741 (2013).</li><li>Arai, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+cell+of+origin+and+propagation+potential+of+the+fibroblast+growth+factor+9-induced+mouse+model+of+lung+adenocarcinoma.">Characterization of the cell of origin and propagation potential of the fibroblast growth factor 9-induced mouse model of lung adenocarcinoma.</a> J. Pathol. 235, 593-605 (2015).</li><li>Curtis, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+tumor+genotype+is+an+important+determinant+in+identification+of+lung+cancer+propagating+cells.">Primary tumor genotype is an important determinant in identification of lung cancer propagating cells.</a> Cell Stem Cell. 7, 127-133 (2010).</li><li>Lau, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-propagating+cells+and+Yap/Taz+activity+contribute+to+lung+tumor+progression+and+metastasis.">Tumor-propagating cells and Yap/Taz activity contribute to lung tumor progression and metastasis.</a> EMBO J. 33, 468-481 (2014).</li><li>Santos, A. M., Jung, J., Aziz, N., Kissil, J. L., Pur&#233;, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+fibroblast+activation+protein+inhibits+tumor+stromagenesis+and+growth+in+mice.">Targeting fibroblast activation protein inhibits tumor stromagenesis and growth in mice.</a> J Clin Invest. 119, 3613-3625 (2009).</li><li>Zinn, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+Bioluminescence+Imaging+in+Small+Animals.">Noninvasive Bioluminescence Imaging in Small Animals.</a> ILAR J. 49, 103-115 (2008).</li><li>Yao, R., Lecomte, R., Crawford, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-Animal+PET:+What+Is+It,+and+Why+Do+We+Need+It.">Small-Animal PET: What Is It, and Why Do We Need It.</a> J Nucl Med Technol. 40, 157-165 (2012).</li><li>Haruyama, N., Cho, A., Kulkarni, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview:+Engineering+transgenic+constructs+and+mice.">Overview: Engineering transgenic constructs and mice.</a> Curr Protoc Cell Biol. , (2009).</li></ol>

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...

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。

<table><tbody><tr><td>micro-X-ray&amp;ndash;computed tomography</td><td>Rigaku</td><td>R_mCT2</td><td></td></tr><tr><td>NanoZoomer RS Digital Pathology System</td><td>Hamamatsu&amp;nbsp;</td><td>RS C10730</td><td></td></tr><tr><td>NDP.view2&amp;nbsp;Viewing software</td><td>Hamamatsu&amp;nbsp;</td><td>U12388-01</td><td>http://www.hamamatsu.com/jp/en/U12388-01.html</td></tr><tr><td>Isoflurane Vaporizer - Funnel-Fill</td><td>VETEQUIP</td><td>911103</td><td></td></tr><tr><td>Induction chamber, 2 L&amp;nbsp; W9.5 &amp;times; D23 &amp;times; H9.5</td><td>VETEQUIP</td><td>941444</td><td></td></tr><tr><td>Isoflurane</td><td>Mylan</td><td>ES2303-01</td><td></td></tr><tr><td>AZD 4547</td><td>LC Labratories</td><td>A-1088</td><td></td></tr><tr><td>Pentobarbital</td><td>Kyoritsu</td><td>SOM02-YA1312</td><td></td></tr><tr><td>G24 cannula&amp;nbsp;</td><td>Terumo</td><td>SP-FS2419</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Wako</td><td>163-20145</td><td></td></tr><tr><td>Microtome</td><td>Leica</td><td>RM2265</td><td></td></tr><tr><td>Doxycycline</td><td>SLC Japan/PMI Nutrition International</td><td>5TP7</td><td></td></tr><tr><td>ImageJ software&amp;nbsp;</td><td>National Institute of health</td><td></td><td>http://imagej.nih.gov/ij/</td></tr><tr><td>Puralube vet ointment (Occular lubricant)</td><td>Dechra</td><td>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?...

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

Research

JoVE Journal

Medicine

10.7K 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

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.

生物发光和微型CT成像监测肿瘤转移和溶骨性病变

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

科研

医学

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET) imaging. Co-mutations in the KRAS proto-oncogene and the LKB1 tumor suppressor gene are frequent events in lung cancer that drive hypermetabolic, glycolytic tumor growth. A critical pathway regulating the growth and metabolism of these tumors is the mechanistic target of the rapamycin (mTOR) pathway, which can be effectively targeted using selective catalytic mTOR kinase inhibitors. The mTOR inhibitor MLN0128 suppresses glycolysis in mice bearing tumors with Kras and Lkb1 co-mutations, referred to as KL mice. The therapy response in KL mice is first measured by 18F-FDG PET and computed tomography (CT) imaging before and after the delivery of MLN0128. By utilizing 18F-FDG PET/CT, researchers are able to measure dynamic changes in the glucose metabolism in genetically engineered mouse models (GEMMs) of lung cancer following a therapeutic intervention with targeted therapies. This is followed by ex vivo autoradiography and a quantitative immunohistochemical (qIHC) analysis using morphometric software. The use of qIHC enables the detection and quantification of distinct changes in the biomarker profiles following treatment as well as the characterization of distinct tumor pathologies. The coupling of PET imaging to quantitative histology is an effective strategy to identify metabolic and therapeutic responses in vivo in mouse models of disease.

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

In this protocol, we describe how to utilize [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography (18F-FDG PET/CT) imaging to measure the tumor metabolic response to the targeted therapy MLN0128 in a Kras/Lkb1 mutant mouse model of lung cancer and coupled imaging with high resolution ex vivo autoradiography and quantitative histology.

利用18f-葡萄糖 PET/CT 成像和定量组织学测量肺癌小鼠模型葡萄糖代谢的动态变化

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography ...

癌症研究

Computed Tomography-guided Time-domain Diffuse Fluorescence Tomography in Small Animals for Localization of Cancer Biomarkers

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light absorption by tissue chromophores (e.g., hemoglobin, water, lipids, melanin) typically limits optical signal propagation through thicknesses larger than a few millimeters2. Compared to other visible wavelengths, tissue absorption for red and near-infrared (near-IR) light absorption dramatically decreases and non-elastic scattering becomes the dominant light-tissue interaction mechanism. The relatively recent development of fluorescent agents that absorb and emit light in the near-IR range (600-1000 nm), has driven the development of imaging systems and light propagation models that can achieve whole body three-dimensional imaging in small animals3.
Despite great strides in this area, the ill-posed nature of diffuse fluorescence tomography remains a significant problem for the stability, contrast recovery and spatial resolution of image reconstruction techniques and the optimal approach to FMI in small animals has yet to be agreed on. The majority of research groups have invested in charge-coupled device (CCD)-based systems that provide abundant tissue-sampling but suboptimal sensitivity4-9, while our group and a few others10-13 have pursued systems based on very high sensitivity detectors, that at this time allow dense tissue sampling to be achieved only at the cost of low imaging throughput. Here we demonstrate the methodology for applying single-photon detection technology in a fluorescence tomography system to localize a cancerous brain lesion in a mouse model.
The fluorescence tomography (FT) system employed single photon counting using photomultiplier tubes (PMT) and information-rich time-domain light detection in a non-contact conformation11. This provides a simultaneous collection of transmitted excitation and emission light, and includes automatic fluorescence excitation exposure control14, laser referencing, and co-registration with a small animal computed tomography (microCT) system15. A nude mouse model was used for imaging. The animal was inoculated orthotopically with a human glioma cell line (U251) in the left cerebral hemisphere and imaged 2 weeks later. The tumor was made to fluoresce by injecting a fluorescent tracer, IRDye 800CW-EGF (LI-COR Biosciences, Lincoln, NE) targeted to epidermal growth factor receptor, a cell membrane protein known to be overexpressed in the U251 tumor line and many other cancers18. A second, untargeted fluorescent tracer, Alexa Fluor 647 (Life Technologies, Grand Island, NY) was also injected to account for non-receptor mediated effects on the uptake of the targeted tracers to provide a means of quantifying tracer binding and receptor availability/density27. A CT-guided, time-domain algorithm was used to reconstruct the location of both fluorescent tracers (i.e., the location of the tumor) in the mouse brain and their ability to localize the tumor was verified by contrast-enhanced magnetic resonance imaging.
Though demonstrated for fluorescence imaging in a glioma mouse model, the methodology presented in this video can be extended to different tumor models in various small animal models potentially up to the size of a rat17.

Diffuse fluorescence tomography offers a relatively low-cost and potentially high-throughout approach to preclinical in vivo tumor imaging. The methodology of optical data collection, calibration, and image reconstruction is presented for a computed tomography-guided non-contact time-domain system using fluorescent targeting of the tumor biomarker epidermal growth factor receptor in a mouse glioma model.

电脑断层导引的时域漫荧光体层摄影术在小动物的肿瘤生物标志物的本土化

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

实时成像在乳腺癌小鼠模型中的肿瘤微环境对药物的反应

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic alterations in oncogenes such as the KRAS oncogene, which constitutes ~25% of lung cancer cases. The difficulty in therapeutically targeting KRAS-driven lung cancer partly stems from having poor models that can mimic the progression of the disease in the lab. We describe a method that permits the relative quantification of primary KRAS lung tumors in a Cre-inducible LSL-KRAS G12D mouse model via ultrasound imaging. This method relies on brightness (B)-mode acquisition of the lung parenchyma. Tumors that are initially formed in this model are visualized as B-lines and can be quantified by counting the number of B-lines present in the acquired images. These would represent the relative tumor number formed on the surface of the mouse lung. As the formed tumors develop with time, they are perceived as deep clefts within the lung parenchyma. Since the circumference of the formed tumor is well-defined, calculating the relative tumor volume is achieved by measuring the length and width of the tumor and applying them in the formula used for tumor caliper measurements. Ultrasound imaging is a non-invasive, fast and user-friendly technique that is often used for tumor quantifications in mice. Although artifacts may appear when obtaining ultrasound images, it has been shown that this imaging technique is more advantageous for tumor quantifications in mice compared to other imaging techniques such as computed tomography (CT) imaging and bioluminescence imaging (BLI). Researchers can investigate novel therapeutic targets using this technique by comparing lung tumor initiation and progression between different groups of mice.

This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.

超声成像对小鼠肺肿瘤进展的检测

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...

High-Resolution Ultrasonography for the Analysis of Orthotopic ATC Tumors in a Genetically Engineered Mouse Model

Anaplastic thyroid carcinoma (ATC) is associated with a poor prognosis and short median survival time, but no effective treatment improves the outcomes significantly. Genetically engineered murine models that mimic ATC&#39;s progression may help researchers to study treatments for this disease. Crossing three different genotypes of mice, a TPO-cre/ERT2; BrafCA/wt; Trp53&#916;ex2-10/&#916;ex2-10&#160;transgenic ATC model was developed. The ATC murine model was induced by an intraperitoneal injection of tamoxifen with overexpression of BrafV600E and deletion of Trp53, and the tumors were generated within about 1 month. High-resolution ultrasound was applied to investigate the tumor initiation and progression, and the dynamic growth curve was obtained by measuring the tumor sizes. Compared to magnetic resonance imaging (MRI) and computed tomography scanning, ultrasound has advantages in observing the ATC murine model, such as being noninvasive, portable, in real-time, and without radiation exposure. High-resolution ultrasound is suitable for dynamic and multiple measurements. However, ultrasonographic examination of the thyroid in mice requires relevant anatomical knowledge and experience. This article provides a detailed procedure for utilizing high-resolution ultrasound to scan tumors in the transgenic ATC model. Meanwhile, ultrasonic parameter adjustment, ultrasound scanning skills, anesthesia and recovery of the animals, and other elements that need attention during the process are listed.

The present protocol describes high-frequency ultrasonography for visualizing the entire mouse thyroid gland and monitoring the growth of anaplastic thyroid carcinoma.

用于分析基因工程小鼠模型中原位ATC肿瘤的高分辨率超声检查

Anaplastic thyroid carcinoma (ATC) is associated with a poor prognosis and short median survival time, but no effective treatment improves the outcomes ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

评价肺转移的小鼠乳腺肿瘤模型定量实时荧光定量PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

A Multimodal Imaging Approach Based on Micro-CT and Fluorescence Molecular Tomography for Longitudinal Assessment of Bleomycin-Induced Lung Fibrosis in Mice

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which causes significant deterioration in lung function and subsequent death from respiratory failure.
The pathogenesis of IPF in experimental animal models has been induced by bleomycin administration. In this study, we investigate an IPF-like mouse model induced by a double intratracheal bleomycin instillation. Standard histological assessments used for studying lung fibrosis are invasive terminal procedures. The goal of this work is to monitor lung fibrosis through noninvasive imaging techniques such as Fluorescent Molecular Tomography (FMT) and Micro-CT. These two technologies validated with histology findings could represent a revolutionary functional approach for real time non-invasive monitoring of IPF disease severity and progression. The fusion of different approaches represents a step further for understanding the IPF disease, where the molecular events occurring in a pathological condition can be observed with FMT and the subsequent anatomical changes can be monitored by Micro-CT.

We describe a non-invasive multimodal imaging approach based on Micro-CT and fluorescence molecular tomography for longitudinal assessment of the mouse lung fibrosis model induced by double intratracheal instillation of bleomycin.

基于显微 CT 和荧光分子层析成像的多模态成像方法对博莱霉素诱导的小鼠肺纤维化的纵向评价

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which ...

免疫与感染

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

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. MPM development is mainly associated with asbestos. Treatments provide only modest survival since the median survival average is 9&#8211;18 months from the time of diagnosis. Therefore, more effective treatments must be identified. Most data describing new therapeutic targets were obtained from in vitro experiments and need to be validated in reliable in vivo preclinical models. This article describes one such reliable MPM orthotopic model obtained after injection of a human MPM cell line H2052/484 into the pleural cavity of immunodeficient athymic mice. Transplantation in the orthotopic site allows studying the progression of tumor in the natural in vivo environment. Positron emission tomography/computed tomography (PET/CT) molecular imaging using the clinical [18F]-2-fluoro-2-deoxy-D-glucose ([18F]FDG) radiotracer is the diagnosis method of choice for examining patients with MPM. Accordingly, [18F]FDG-PET/CT was used to longitudinally monitor the disease progression of the H2052/484 orthotopic model. This technique has a high 3R potential (Reduce the number of animals, Refine to lessen pain and discomfort, and Replace animal experimentation with alternatives) since the tumor development can be monitored non-invasively and the number of animals required could be significantly reduced.
This model displays a high development rate, a rapid tumor growth, is cost-efficient and allows for rapid clinical translation. By using this orthotopic xenograft MPM model, researchers can assess biological responses of a reliable MPM model following therapeutic interventions.

This article describes the generation of an orthotopic mouse model of human pleural mesothelioma by implantation of H2052/484 mesothelioma cells into the pleural cavity of immunocompromised athymic mice. The longitudinal monitoring of the development of intrapleural tumors was assessed by non-invasive multimodal [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography imaging.

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. ...

In vivo Evaluation of Mucociliary Clearance in Mice

Respiratory motile cilia, specialized organelles of the cell, line the apical surface of epithelial cells lining the respiratory tract. By beating in a metachronal, synchronal fashion, these multiple, motile, actin-based organelles generate a cephalad fluid flow clearing the respiratory tract of inhaled pollutants and pathogens. With increasing environmental pollution, novel viral pathogens and emerging multi-drug resistant bacteria, cilia generated mucociliary clearance (MCC) is essential for maintaining lung health. MCC is also depressed in multiple congenital disorders like primary ciliary dyskinesia, cystic fibrosis as well as acquired disorders like chronic obstructive pulmonary disease. All these disorders have established, in some case multiple, mouse models. In this publication, we detail a method using a small amount of radioactivity and dual-modality SPECT/CT imaging to accurately and reproducibly measure MCC in mice in vivo. The method allows for recovery of mice after imaging, making serial measurements possible, and testing potential therapeutics longitudinally over time. The data in wild-type mice demonstrates the reproducibility of the MCC measurement as long as adequate attention to detail is paid, and the protocol strictly adhered to.

In this publication, we describe protocols for assessing airway mucociliary clearance (MCC) in mice in vivo utilizing dual-modality radionuclide imaging. This protocol is designed for a single photon emission computed tomography (SPECT) and computed tomography (CT) acquisition protocol using mouse whole body (MWB) collimators in a dual SPECT/CT system.

在体内评估小鼠的粘液清除

Respiratory motile cilia, specialized organelles of the cell, line the apical surface of epithelial cells lining the respiratory tract. By beating in a ...