这项研究由德意志 Krebshilfe (最大 Eder 小组到: 110972) 支持, DGVS 博士论文奖学金 (对 SMB) 和其他 Kröner 费森尤斯基础奖学金 (对 RGG) 在大学医疗中心格廷根。我们感谢 Jutta 布伦伯格和 Ulrike 韦格纳专家的技术援助。我们还感谢所有动物技术员在大学医学中心的动物设施格廷根为老鼠保存。所有的实验都是根据德国动物福利条例进行的。

这个协议是根据动物关心指南在大学医疗中心格廷根, 德国 (33.9-42502-04-15/2056)。根据个别动物评审委员会的具体要求, 一些协议步骤可以相应地加以修改。
1. 科索沃保护团小鼠的腹部触诊
<ol><li>为了避免不必要的超声扫描, 触及小鼠腹部, 以确定可能有腹腔病变的小鼠, 并随后接受腹部超声检查。<ol>
<li>每周从8周开始, 在 KPC 的小鼠模型中进行腹部触诊。高分辨率超声也可以应用于移植的肿瘤模型 (例如, orthotopics) 或其他基因工程老鼠模型。 注: 根据肿瘤的动力学和研究目标 (预防研究, 治疗干预研究), 检测非常小的肿瘤 (1-2 毫米) 是可行的, 以监测肿瘤发病前肿瘤可以触及。</li>
	</ol>
</li>
	<li>将鼠标放在笼栅格上, 然后用不固定手的索引和拇指轻轻地上下移动鼠标, 并触及腹部 (图 1)。</li>
	<li>如果鼠标耐受软触诊, 继续缓慢增加压力。更大的抵抗可以很容易地被发现在上腹部和下腹。有时, 硬粪可以被错误地解释为肿瘤肿块。</li>
</ol>2. 工作空间的准备
<ol><li>为至少三个不同的工作单元提供空间: 预扫描区域、扫描阶段和恢复笼。所需的材料在图 2中进行了描述。</li>
	<li>清洁主工作板 (例如70% 乙醇), 并在扫描前打开加热平台 (图 3)。确保在第一个鼠标转移之前达到38-40 °c 的目标温度。</li>
	<li>打开超声波机, 并为获取的数据创建一个新文件夹 (图 4)。B 模式设置用于腹部成像。另外, 确保将腹式换能器插入机器的主动端。使用传感器安装系统将传感器头固定在工作台上的顶部。</li>
	<li>在加热板或热垫上准备一个恢复笼 (或使用加热灯), 以确保扫描后的小鼠的程序体温和快速恢复。在这个笼子的底部放一层薄的纸巾作为垫子, 以避免床上有窒息的可能 (图 5)。</li>
</ol>3. 吸入麻醉
<ol><li>将鼠标放入感应腔内进行操作。打开气体载波流阀 (1 升/分氧或空气), 并将异氟醚浓度设置为 4%, 使感应腔的饱和度逐渐增加。请确保适当的异氟醚清除 (被动或主动过滤) 到位。大约2-4 分钟后, 老鼠通常会在深镇静下。<ol>
<li>在工作台上通过鼻锥管理2% 异氟醚, 保持镇静。</li>
	</ol>
</li>
	<li>小心地将动物从感应室转移到工作台上。检查鼻锥是否有恒定的气流。把鼠标的鼻子放在麻醉线出口的鼻锥上。初始扫描的常规位置为仰卧位。鼠标也可以放置在其右侧或左侧, 以提高胰头或尾部肿瘤的可见性 (图 6)。</li>
	<li>虽然腹部超声不是痛苦的本身, 执行脚捏测试作为一个指标的疼痛自由状态和深层麻醉状态. 如果老鼠仍然活跃或活跃地移动, 转让动物回到诱导室为反复和延长的诱导。</li>
	<li>使用眼部保护软膏, 防止因液体流失引起的角膜损伤。</li>
	<li>在麻醉过程中, 使用监控系统。然而, 心电图和直肠温度不定期用于腹部超声扫描。重要的是, 老鼠失去了在麻醉下 thermoregulate 的能力。因此, 密切观察临床症状的体温过低或呼吸窘迫, 如喘息或窒息。如果任何症状明显停止异氟醚, 并立即将鼠标放在恢复笼中。</li>
</ol>4. 超声波设置
<ol><li>根据制造商的说明, 打开超声波系统。确保将主要设置放在常规成像与腹部包裹激活。通常, B 模式成像被用作数据获取的标准。</li>
	<li>通过以下参数实现优化的图像质量: 频率40兆赫, 2 d 增益 25-30 dB, 图像深度10毫米, 图像宽度10毫米, 3 焦带中心在3-6 毫米之间。</li>
</ol>5. 鼠标准备
<ol><li>用胶带固定上、下下肢。尽可能小心, 以避免对动物的任何伤害, 由于固定 (图 7)。</li>
	<li>使用电子宠物剪刀去除所有腹部毛皮。由于毛皮质地, 从下腹部开始到胸腔。在突 xiphoideus中保重, 以避免此区域的皮肤损伤。 注: 鼠标皮毛反射超声波, 降低能见度。</li>
	<li>使用脱毛霜进行优化的皮肤准备和彻底去除毛皮。使用棉花尖 (图 8) 在剃掉的腹部上涂一层薄薄的奶油。除去奶油 (三十年代以后或以后取决于使用的奶油) 与干燥组织然后充足的水完全地去除所有残余 (图 9,图 10,图 11)。奶油残余可能导致皮肤和粘膜刺激后, 超声, 并可能因此影响健康。</li>
	<li>为了确保充分的扫描条件, 应用大量的超声波凝胶到腹部 (图 12)。通常, 使用温热超声凝胶, 以最大限度地减少对小鼠的体温过低。</li>
	<li>在传感器头上手动应用一层薄薄的超声波凝胶。通过触摸传感器端的凹槽来执行测试。在屏幕上, 如果传感器正确定位, 则可以看到左上侧的信号。</li>
</ol>6. 腹部超声
<ol><li>使用牢固的压力将传感器调整到腹部。大鼠腹部的压迫可能导致呼吸或循环损伤, 应该避免。</li>
	<li>向上和向下扫描。调整工作平台的框架和 x 轴和 y 轴的车轮。</li>
	<li>作为第一步确定两个肾脏。通常他们在下腹部出现作为 hyperechogenic 均匀结构。连接的腔 renalis可以很容易地找到, 进一步导致腔静脉下 (图 13)。</li>
	<li>使用腔 lienalis作为胰腺的标志性容器。尝试区分腔 renalis和腔 lienalis。在检测到腔 lienalis时, 胰腺可以完全定位。正常的胰腺, 而不是一个坚实的结构 (图 14 和图 15) 通过腹腔传播的膜。</li>
</ol>7. 胰腺肿瘤的检测和容积评估
<ol><li>识别所有周围结构的胰腺病变。它们通常显示为回声非均匀圆形或拉长的组织肿块 (图 16)。肿瘤通常是牢固的, 并且不可能由扫描头压缩。注意, 不时地区分肿瘤和小肠循环可能是很棘手的。注意在胰腺肿瘤中不应观察到的肠道蠕动。</li>
	<li>在肿瘤检测屏幕上向上和向下, 以获得总大小的印象和测量最大直径使用超声波钳。一旦找到合适的图像, 冻结屏幕, 并使用测量工具来精确确定肿瘤的大小。注意, 为以后使用它将是重要的区分肿瘤是否在胰腺的尾巴、身体或头被定位。</li>
	<li>在更改回实时模式之前, 请确保保存图片以记录工作。注意, 在冻结图像时, 可能会发生调整轮被释放, 而超声波探头甚至更压在鼠标腹部。因此, 在将模式更改为冻结之前, 请修复压缩的调整。</li>
	<li>完成第一个平面的测量后, 可以在纵向位置 (图 17) 旋转扫描头, 或者将鼠标转到其侧面, 以检测肿瘤的纵向扩张。有些肿瘤可能需要改变鼠标的位置以获得足够的能见度。对于胰尾肿瘤, 鼠标可以放在右侧 (图 10 和图 19), 用于左侧的胰头肿瘤 (图 11 和图 20)。</li>
</ol>8. 肿瘤体积的量化
注意: 所有努力的一个主要目的是正确确定肿瘤体积。虽然有几个技术可利用一个演算方法包括椭球的惯例在大学医疗中心格廷根是优选的。
<ol><li>配备所有三以前获得的直径, 计算相应肿瘤的近似体积 (图 21)。</li>
	<li>或者, 使用扫描头的马达系统对肿瘤区域进行自动扫描, 随后进行体积计算和3维重建。</li>
</ol>9. 恢复
<ol><li>扫描后, 小心取出所有胶带, 并仔细删除所有超声凝胶与干燥组织。</li>
	<li>小心地将鼠标转移到恢复笼中 (图 21)。避免 mouse´s 脸上的任何覆盖组织, 可能会导致呼吸障碍。应首选恢复的侧面位置。</li>
</ol>

<ol><li>Kersten, K., de Visser, K. E., van Miltenburg, M. H., Jonkers, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetically+engineered+mouse+models+in+oncology+research+and+cancer+medicine%5BTitle%5D)+AND+%22EMBO+Molecular+Medicine%22%5BJournal%5D)">Genetically engineered mouse models in oncology research and cancer medicine.</a> EMBO Molecular Medicine. 9 (2), 137-153 (2017).</li>
<li>Olive, K. P., Politi, K. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aOlive%2C+KP+%22Translational+therapeutics+in+genetically+engineered+mouse+models+of+cancer%22">Translational therapeutics in genetically engineered mouse models of cancer</a>. (2), Cold Spring Harbor. 131-143 (2014).</li>
<li>Westphalen, C. B., Olive, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetically+engineered+mouse+models+of+pancreatic+cancer%5BTitle%5D)+AND+%22The+Cancer+Journal%22%5BJournal%5D)">Genetically engineered mouse models of pancreatic cancer.</a> The Cancer Journal. 18 (6), 502-510 (2012).</li>
<li>Hingorani, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Trp53R172H+and+KrasG12D+cooperate+to+promote+chromosomal+instability+and+widely+metastatic+pancreatic+ductal+adenocarcinoma+in+mice%5BTitle%5D)+AND+%22Cancer+Cell%22%5BJournal%5D)">Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice.</a> Cancer Cell. 7 (5), 469-483 (2005).</li>
<li>Frese, K. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((nab-Paclitaxel+potentiates+gemcitabine+activity+by+reducing+cytidine+deaminase+levels+in+a+mouse+model+of+pancreatic+cancer%5BTitle%5D)+AND+%22Cancer+Discovery%22%5BJournal%5D)">nab-Paclitaxel potentiates gemcitabine activity by reducing cytidine deaminase levels in a mouse model of pancreatic cancer.</a> Cancer Discovery. 2 (3), 260-269 (2012).</li>
<li>Paredes, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+non-invasive+method+of+quantifying+pancreatic+volume+in+mice+using+micro-MRI%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">A non-invasive method of quantifying pancreatic volume in mice using micro-MRI.</a> PLoS One. 9 (3), e92263(2014).</li>
<li>Boj, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Organoid+models+of+human+and+mouse+ductal+pancreatic+cancer%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Organoid models of human and mouse ductal pancreatic cancer.</a> Cell. 160 (1-2), 324-338 (2015).</li>
<li>Aung, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunotargeting+of+Integrin+alpha6beta4+for+Single-Photon+Emission+Computed+Tomography+and+Near-Infrared+Fluorescence+Imaging+in+a+Pancreatic+Cancer+Model%5BTitle%5D)+AND+%22Molecular+Imaging%22%5BJournal%5D)">Immunotargeting of Integrin alpha6beta4 for Single-Photon Emission Computed Tomography and Near-Infrared Fluorescence Imaging in a Pancreatic Cancer Model.</a> Molecular Imaging. 15, (2016).</li>
<li>Akladios, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Contribution+of+microCT+structural+imaging+to+preclinical+evaluation+of+hepatocellular+carcinoma+chemotherapeutics+on+orthotopic+graft+in+ACI+rats%5BTitle%5D)+AND+%22Bulletin+du+Cancer%22%5BJournal%5D)">Contribution of microCT structural imaging to preclinical evaluation of hepatocellular carcinoma chemotherapeutics on orthotopic graft in ACI rats.</a> Bulletin du Cancer. 98 (2), 120-132 (2011).</li>
<li>Neesse, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CTGF+antagonism+with+mAb+FG-3019+enhances+chemotherapy+response+without+increasing+drug+delivery+in+murine+ductal+pancreas+cancer%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Science+USA%22%5BJournal%5D)">CTGF antagonism with mAb FG-3019 enhances chemotherapy response without increasing drug delivery in murine ductal pancreas cancer.</a> Proceedings of the National Academy of Science USA. 110 (30), 12325-12330 (2013).</li>
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</ol>

作者没有什么可透露的。

通过该协议, 给出了在基因工程小鼠模型中应用高分辨率腹部超声显像对胰腺肿瘤进行量化的详细描述。最近, Sastra et . 发表了一个详细的描述如何量化的胰腺肿瘤的小鼠模型, 但没有可视化的指示, 关于准备和处理作为先决条件的所有进一步步骤显示11。本手稿的总体目标是为小鼠的高分辨率超声提供一个全面的视觉指导。
虽然我们对基因工程小鼠内?...

基因工程鼠模型在癌症研究中得到了越来越重要的地位, 因为它们能够密切重述人类致癌的复杂性质1,2,3。研究胰腺癌发展、进展和治疗反应的最常用模型之一是 Kras 癌基因的活化突变和抑癌 p534的灭活。此LSL KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (KPC) 小鼠模型模拟了从前侵袭性胰腺上皮内肿瘤 (PanIN) 病变到浸润性癌的步进式进展。表型, 几乎所有的老鼠在出生后的头六月内发育 PDAC。然而, 与移植模型相比, KPC 模型揭示了一个高度可变的肿瘤发病时间从8周到几个月的4。一旦胰腺肿瘤达到一定的大小 (直径5-9 毫米), 肿瘤生长迅速加速, 小鼠将不得不在临床前试验中注册5。因此, 准确检测肿瘤发病和肿瘤大小是临床前期研究后勤和治疗监测的必要前提。一般而言, 几种方法如磁共振成像 (MRI)6, 计算机断层扫描7,8,9或高分辨率超声可用于进行肿瘤筛查和治疗10。每种技术都有其优点和缺点。虽然 MRI 或计算机断层扫描 (CT) 成像允许高分辨率的数据采集和准确的体积计算, 在一般镇静下延长检查时间和非常昂贵的设备是必需的, 不允许频繁扫描了很长一段时间。相比之下, 小动物超声是一种已建立的方法, 可用于筛查小鼠的腹部病理11。这种成像方法的优点是扫描时间短, 分辨率高, 并且有可能使用多普勒超声或造影增强超声 (CEUS) 来可视化器官的灌注。然而, 正确的图像解释需要解剖学知识、3D 想象力和彻底的实践训练。
本文给出了在 KPC 模型中利用高分辨率超声的详细协议。此外, 标准的超声图像被描绘和标签的器官结构, 以促进方向的调查。

<table><tbody><tr><td>Visual Sonics Vevo2100 High Resolution Ultrasound System, including imaging stage and anesthesia line</td><td>FUJIFILM VisualSonics Inc, Canada</td><td>VS-11945</td><td></td></tr><tr><td>Vevo 2100 MicroScan Transducer MS-550-D (22-55MHz)</td><td>FUJIFILM VisualSonics Inc, Canada</td><td>VS-11874</td><td></td></tr><tr><td>Vevo Anesthesia System (anesthesia induction chamber with fresh and waste gas inlet)</td><td>FUJIFILM VisualSonics Inc, Canada</td><td>SA-12055</td><td></td></tr><tr><td>Vevo Imaging Station (working stage with nose cone for anesthesia supply)</td><td>FUJIFILM VisualSonics Inc, Canada</td><td>SA-11982</td><td></td></tr><tr><td>&amp;nbsp;electronic pet clippers</td><td>Panasonic Marketing Europe, Germany</td><td>5025232484324</td><td>Panasonic ER-PA10-s</td></tr><tr><td>Labotect Hot plate</td><td>Labor tech G&amp;ouml;ttingen, Germany</td><td>13854</td><td></td></tr><tr><td>eye cream (ophthalmic ointment)</td><td>Sch&amp;uuml;lke&amp;amp;Mayr, Germany</td><td>9080249</td><td></td></tr><tr><td>veterinary isoflurane</td><td>Abbvie, Germany</td><td>4831867</td><td></td></tr><tr><td>depilatory cream</td><td>RB healthcare UK, United Kingdom</td><td>8218535</td><td></td></tr><tr><td>70% ethanol (v/v) in distilled water</td><td>TH. Geyer, Germany</td><td>22941000</td><td></td></tr><tr><td>ultrasound gel</td><td>Asmuth, Germany</td><td>13477</td><td></td></tr><tr><td>tissue wipes</td><td>Kimberly-Clark Germany, Germany</td><td>7558</td><td></td></tr><tr><td>cotton tips</td><td>Meditrade, Germany</td><td>75481116</td><td></td></tr><tr><td>glass bowl for ultrasound gel</td><td>ARC France, France</td><td>H1149</td><td></td></tr><tr><td>water bowl</td><td>W &amp;amp; P Trading Co., USA</td><td>B00K2P6PLQ</td><td></td></tr><tr><td>gauze sponges</td><td>Fuhrmann, Germany</td><td>960504</td><td></td></tr></tbody></table>

utilizing high resolution ultrasound to monitor tumor onset and growth in genetically engineered pancreatic cancer models

LSL KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (KPC) 小鼠模型代表了一种建立和常用的转基因模型来评价胰腺癌的新疗法。在8周和几个月间, 科索沃保护团模型中的肿瘤发病是可变的。因此, 非侵入性成像工具需要筛查肿瘤发病和监测反应治疗。为解决这一问题, 在过去几年中出现了各种不同的办法。高分辨率超声具有无侵袭、速度快、图像分辨率高、无辐射照射等优点。然而, 超声在小鼠是不琐碎的, 充分的解剖学知识和实际技能, 需要成功地执行高分辨率超声在前胰腺癌模型。在下面的文章中, 详细介绍了腹部超声在小鼠模型中的具体操作指南, 特别着重于内源性胰腺癌模型。此外, 还提供了常见的错误和如何避免它们的摘要。

超声成像是一种多功能和非侵入性的技术, 用于解决小鼠模型的人类疾病的几个问题。与所有其他成像方法相比, 主要优点是高通量、成本效率高、采集时间短和实时成像。但是, 此工具需要专门知识才能生成准确、高质量的图像。特别是在不需要的文物的情况下, 至少有一些经验, 超声成像一般是非常有用的。关于胰腺癌, 这个工具允许确定肿瘤的发病、进展和对治疗的反?...

Watch this Scientific Journal Video about Utilizing High Resolution Ultrasound to Monitor Tumor Onset and Growth in Genetically Engineered Pancreatic Cancer Models at JoVE.com

Utilizing High Resolution Ultrasound to Monitor Tumor Onset and Growth in Genetically Engineered Pancreatic Cancer Models

本文介绍了高分辨率超声在基因工程胰腺癌小鼠中的应用。其主要目的是为内源性胰腺肿瘤的检测和评价提供详细的指导。

应用高分辨率超声监测基因工程胰腺癌的肿瘤发病和生长

The LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mouse model represents an established and frequently used transgenic model to evaluate novel ...

utilizing-high-resolution-ultrasound-to-monitor-tumor-onset-growth

Research

JoVE Journal

Cancer Research

10.8K 次观看.  University Medical Center Goettingen. 本文介绍了高分辨率超声在基因工程胰腺癌小鼠中的应用。其主要目的是为内源性胰腺肿瘤的检测和评价提供详细的指导。

视频: Utilizing High Resolution Ultrasound to Monitor Tumor Onset and Growth in Genetically Engineered Pancreatic Cancer Models

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

胰腺癌的荧光原位小鼠模型

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

科研

癌症研究

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 ...

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 ...

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

生物荧光原位模型胰腺癌进展

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

医学

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 ...

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 ...

使用植入式稳定窗的小鼠胰腺成像

Stabilized Window for Intravital Imaging of the Murine Pancreas

The physiology and pathophysiology of the pancreas are complex. Diseases of the pancreas, such as pancreatitis and pancreatic adenocarcinoma (PDAC) have high morbidity and mortality. Intravital imaging (IVI) is a powerful technique enabling the high-resolution imaging of tissues in both healthy and diseased states, allowing for real-time observation of cell dynamics. IVI of the murine pancreas presents significant challenges due to the deep visceral and compliant nature of the organ, which make it highly prone to damage and motion artifacts. 
Described here is the process of implantation of the Stabilized Window for Intravital imaging of the murine Pancreas (SWIP). The SWIP allows IVI of the murine pancreas in normal healthy states, during the transformation from the healthy pancreas to acute pancreatitis induced by cerulein, and in malignant states such as pancreatic tumors. In conjunction with genetically labeled cells or the administration of fluorescent dyes, the SWIP enables the measurement of single-cell and subcellular dynamics (including single-cell and collective migration) as well as serial imaging of the same region of interest over multiple days. 
The ability to capture tumor cell migration is of particular importance as the primary cause of cancer-related mortality in PDAC is the overwhelming metastatic burden. Understanding the physiological dynamics of metastasis in PDAC is a critical unmet need and crucial for improving patient prognosis. Overall, the SWIP provides improved imaging stability and expands the application of IVI in the healthy pancreas and malignant pancreas diseases.

作者聚焦:通过先进的活体成像彻底改变胰腺疾病的理解 

We present a protocol for the surgical implantation of a stabilized indwelling optical window for subcellular-resolution imaging of the murine pancreas, allowing serial and longitudinal studies of the healthy and diseased pancreas.

用于小鼠胰腺活体成像的稳定窗口

The physiology and pathophysiology of the pancreas are complex. Diseases of the pancreas, such as pancreatitis and pancreatic adenocarcinoma (PDAC) have ...

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.

应用高分辨率超声监测基因工程胰腺癌的肿瘤发病和生长

摘要

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