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>

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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源性胰腺肿瘤的描述, 但这种方法也可以用于原位移植模型。对于原位模型, 即使是非常小的肿瘤高达1-2 毫米可以检测 abdominally 的服用率和肿瘤发病。由于腹部触诊可能是主观的和难以重现, 超声检查也可以应用在没有事先触诊的 KPC 模型, 以检测非常小的胰腺肿瘤的发病。此外, 腹部结构, 如充满空气的小肠循环可以叠加。在这方面, 改变动物在工作台上的位置可能会有帮助。大多数肿瘤可以在仰卧位上可视化;然而, 有些肿瘤只有当鼠标放在左侧 (胰头肿瘤) 或右侧 (胰尾肿瘤) 时才会被检测到。因此, 足够的鼠标定位对于提高有效的能见度至关重要。
没有足够的可见性腹部器官或解剖变异性可能发生。为了提高超声图像的可见度和分辨率, 亮度和对比度设置以及焦点应该相应地改变。图像文物可能是由于缺陷/错误探针, 不完全去除小鼠皮毛, 不足的超声凝胶或接触压力不足的探针与小鼠腹部。此外, 轻微倾斜探头也可以大大提高能见度。
此外, 小动物超声可以对肿瘤生长动力学进行纵向监测。该技术的主要优点包括低成本 (考虑到超声波机的可用性), 没有辐射照射, 扫描时间短, 因此对小鼠的应变小。特别是在纵向癌症研究的基础上, 反复扫描是很容易执行的, 对动物不需要长时间的镇静或麻醉, 这是小动物 CT 或 MRI 扫描所必需的。
由于技术进步与更加强有力的超声系统当前正在开发以改进的分辨率和越来越自动的图像优化工具, 小动物, 高分辨率超声将更经常使用在不同的临床前学科的未来。该协议中的关键步骤包括正确的鼠标准备和定位, 以及正确识别腹部器官和血管。
高分辨率超声是在基因工程和原位小鼠模型中检测和定量胰腺肿瘤的重要工具。与其他已建立的想象技术相比, 这种基于超声的方法结合了快速会话时间和成本效率。由于超声图像的正确采集和解释需要适当的动手训练和经验, 此视频协议作为胰腺癌模型各个方面的详细指南。

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>&#160;electronic pet clippers</td>
			<td>Panasonic Marketing Europe, Germany</td>
			<td>5025232484324</td>
			<td>Panasonic ER-PA10-s</td>
		</tr>
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			<td>Labotect Hot plate</td>
			<td>Labor tech G&#246;ttingen, Germany</td>
			<td>13854</td>
			<td></td>
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			<td>eye cream (ophthalmic ointment)</td>
			<td>Sch&#252;lke&#38;Mayr, Germany</td>
			<td>9080249</td>
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			<td>veterinary isoflurane</td>
			<td>Abbvie, Germany</td>
			<td>4831867</td>
			<td></td>
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		<tr>
			<td>depilatory cream</td>
			<td>RB healthcare UK, United Kingdom</td>
			<td>8218535</td>
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			<td>70% ethanol (v/v) in distilled water</td>
			<td>TH. Geyer, Germany</td>
			<td>22941000</td>
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			<td>ultrasound gel</td>
			<td>Asmuth, Germany</td>
			<td>13477</td>
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			<td>tissue wipes</td>
			<td>Kimberly-Clark Germany, Germany</td>
			<td>7558</td>
			<td></td>
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			<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 &#38; 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、进展和对治疗的反应。此外, 通过测量不同的直径, 它允许精确定量的肿瘤体积随着时间的推移。本研究详细说明和描述了如何使用高分辨率超声成像作为胰腺癌的筛选工具, 在基因工程鼠模型。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56979/56979fig1.jpg"> 图 1: 腹部触诊.固定手轻轻地拉起尾巴, 而非固定手开始过程中轻轻上下移动。<a href="/files/ftp_upload/56979/56979fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56979/56979fig2.jpg"> 图 2: 预先扫描区域, 并概述所有需要的项目.皮肤消毒 (1), 纱布海绵 (2), 70% 异丙醇 (3), 超声凝胶与玻璃肠 (4), 眼科软膏 (5), 纸巾 (6), 医疗胶带 (7), 水容器 (8), 宠物剪 (9) 脱毛奶油与小容器 (10), 棉花提示应用眼膏或超声凝胶 (11)。<a href="/files/ftp_upload/56979/56979fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56979/56979fig3.jpg"> 图 3: 使用异氟醚蒸发器扫描区域.工作台上可调探头架 (1), 感应室 (2), 包括异氟醚蒸发器 (3)。<a href="/files/ftp_upload/56979/56979fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56979/56979fig4.jpg"> 图 4: 超声波设备.超声波系统提供三超声波探头和相应的端口。系统中包括键盘和 PC 鼠标。<a href="/files/ftp_upload/56979/56979fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56979/56979fig5.jpg"> 图 5: 恢复保持架.笼底用薄的组织和预加热的方式准备, 以便在扫描过程后有足够的加热。温度以摄氏 (C°) 显示。<a href="/files/ftp_upload/56979/56979fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56979/56979fig6.jpg"> 图 6: 在工作台上固定鼠标.老鼠在工作台上处于仰卧位。<a href="/files/ftp_upload/56979/56979fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56979/56979fig7.jpg"> 图 7: 使用宠物剪刀后剃光鼠标.腹部毛皮被切开, 但头发的小部分坚持经常导致文物。<a href="/files/ftp_upload/56979/56979fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56979/56979fig8.jpg"> 图 8: 使用脱毛霜.在腹部的剃光区管理一层薄薄的脱毛膏。<a href="/files/ftp_upload/56979/56979fig8large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/56979/56979fig9.jpg"> 图 9: 完全剃掉鼠标的腹部.大量的水被用来冲洗所有的奶油残余物, 只有彻底去除毛皮, 才能获得最佳的扫描程序。<a href="/files/ftp_upload/56979/56979fig9large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/56979/56979fig10.jpg"> 图 10: 鼠标的右侧.胰腺尾部肿瘤的最佳定位。<a href="/files/ftp_upload/56979/56979fig10large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/56979/56979fig11.jpg"> 图 11: 鼠标的左端.胰头肿瘤的最佳定位。<a href="/files/ftp_upload/56979/56979fig11large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/56979/56979fig12.jpg"> 图 12: 鼠标腹部超声探头.为防止对鼠标造成任何伤害, 不要使用太多的压力。<a href="/files/ftp_upload/56979/56979fig12large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 13" class="xfigimg" src="/files/ftp_upload/56979/56979fig13.jpg"> 图 13: 右腹部分的超声图像.主血管结构, 如腹主动脉和腔静脉下等位于接近肾脏和肝脏。<a href="/files/ftp_upload/56979/56979fig13large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 14" class="xfigimg" src="/files/ftp_upload/56979/56979fig14.jpg"> 图 14: 左腹部部分的超声图像.在左肾和脾之间胰腺的膜结构位于, 作为一个地标,腔 lienalis通过器官 (黄色箭头) 运行, 可作为指导结构使用。<a href="/files/ftp_upload/56979/56979fig14large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 15" class="xfigimg" src="/files/ftp_upload/56979/56979fig15.jpg"> 图 15: 胰腺的边框.胃 (与内容) 标记胰腺的左边界 (黄色箭头)。<a href="/files/ftp_upload/56979/56979fig15large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 16" class="xfigimg" src="/files/ftp_upload/56979/56979fig16.jpg"> 图 16: 识别胰腺肿瘤.仰卧位与低密度出现圆形胰腺肿瘤, 箭头指示周围正常组织的边界。<a href="/files/ftp_upload/56979/56979fig16large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 17" class="xfigimg" src="/files/ftp_upload/56979/56979fig17.jpg"> 图 17: 纵向位置.确定胰腺肿瘤的长度。<a href="/files/ftp_upload/56979/56979fig17large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 18" class="xfigimg" src="/files/ftp_upload/56979/56979fig18.jpg"> 图 18: 胰腺肿瘤的纵向扫描.弥漫的十二指肠引起的干扰。<a href="/files/ftp_upload/56979/56979fig18large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 19" class="xfigimg" src="/files/ftp_upload/56979/56979fig19.jpg"> 图 19: 鼠标右端:胰尾肿瘤<a href="/files/ftp_upload/56979/56979fig19large.jpg" target="_blank">请单击此处查看此图的更大版本.</a>
<img alt="Figure 20" class="xfigimg" src="/files/ftp_upload/56979/56979fig20.jpg"> 图 20: 鼠标左端:胰头肿瘤<a href="/files/ftp_upload/56979/56979fig20large.jpg" target="_blank">请单击此处查看此图的更大版本.</a>
<img alt="Figure 21" class="xfigimg" src="/files/ftp_upload/56979/56979fig21.jpg"> 图 21: 肿瘤体积量化.在前述两个扫描位置获得所有三直径后, 进行体积计算。因此, 使用椭球的公式: 4/3 π a/2 * b/2 * c/2。<a href="/files/ftp_upload/56979/56979fig21large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 22" class="xfigimg" src="/files/ftp_upload/56979/56979fig22.jpg"> 图 22: 恢复.扫描后的动物被转移到加热板, 直到完全恢复, 注意到白眼软膏在鼠标的眼睛。<a href="/files/ftp_upload/56979/56979fig22large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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

Nanyang Technological University

Research

JoVE Journal

Cancer Research

10.7K 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

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Genetically engineered mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in large organs such as the colon in which only a single tumor is desired.
As a result, an immunocompetent, genetically engineered mouse model of colorectal cancer was developed which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor development is initiated by surgical, segmental infection of the distal colon with adeno-cre virus in compound conditionally mutant mice. The tumors can be easily detected and monitored via colonoscopy. We here describe the surgical technique of segmental adeno-cre infection of the colon, the surveillance of the tumor via high-resolution colonoscopy and present the resulting colorectal tumors.

A protocol for the establishment of a genetically engineered mouse model of colorectal cancer by segmental adeno-cre infection and its surveillance via high-resolution colonoscopy is presented.

遗传工程小鼠模型的散发性结肠直肠癌

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations ...

科研

癌症研究

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

核素荧光报告基因成像在啮齿动物肿瘤模型中跟踪肿瘤进展

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

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

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

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

患者衍生的人类泌尿细胞癌和结肠直肠癌肿瘤生长和自发转移的正交异种移植模型

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

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ever-increasing number of studies have identified miRNAs as potential biomarkers for cancer diagnosis and prognosis, and also as therapeutic targets, adding an extra dimension to cancer evaluation and treatment. In the context of thyroid cancer, tumorigenesis results not only from mutations in important genes, but also from the overexpression of many miRNAs. Accordingly, the role of miRNAs in the control of thyroid gene expression is evolving as an important mechanism in cancer. Herein, we present a protocol to examine the effects of miRNA-inhibitor delivery as a therapeutic modality in thyroid cancer using human tumor xenograft and orthotopic mouse models. After engineering stable thyroid tumoral cells expressing GFP and luciferase, cells are injected into nude mice to develop tumors, which can be followed by bioluminescence. The in vivo inhibition of a miRNA can reduce tumor growth and upregulate miRNA gene targets. This method can be used to assess the importance of a determined miRNA in vivo, in addition to identifying new therapeutic targets.

This protocol describes xenograft and orthotopic mouse models of human thyroid tumorigenesis as a platform to test microRNA-based inhibitor treatments. This approach is ideal to study the function of non-coding RNAs and their potential as new therapeutic targets.

在微RNA的Vivo抑制下，降低小鼠的肿瘤生长

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ...

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 ...

Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables associated to specific pancreatic pathologies to help preoperative differential diagnosis and patient profiling. Pancreatic juice is a relatively unexplored body fluid, which, due to its close proximity to the tumor site, reflects changes in the surrounding tissue. Here we describe in detail the intraoperative collection procedure. Unfortunately, translating pancreatic juice collection to murine models of PDAC, to perform mechanistic studies, is technically very challenging. Tumor interstitial fluid (TIF) is the extracellular fluid, outside blood and plasma, which bathes tumor and stromal cells. Similarly to pancreatic juice, for its property to collect and concentrate molecules that are found diluted in plasma, TIF can be exploited as an indicator of microenvironmental alterations and as a valuable source of disease-associated biomarkers. Since TIF is not readily accessible, various techniques have been proposed for its isolation. We describe here two simple and technically undemanding methods for its isolation: tissue centrifugation and tissue elution.

Pancreatic juice is a precious source of biomarkers for human pancreatic cancer. We describe here a method for intraoperative collection procedure. To overcome the challenge of adopting this procedure in murine models, we suggest an alternative sample, tumor interstitial fluid, and describe here two protocols for its isolation.

分离近端液研究胰腺腺癌肿瘤微环境

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables ...

A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role of CAFs in the tumor microenvironment can be helpful for understanding the functional importance of fibroblasts, fibroblasts from different tissues, and specific genetic factors in fibroblasts. Mouse models are essential for understanding the contributors to tumor growth and progression in an in vivo context. Here, a protocol in which cancer cells are mixed with fibroblasts and introduced into mice to develop tumors is provided. Tumor sizes over time and final tumor weights are determined and compared among groups. The protocol described can provide more insight into the functional role of CAFs in tumor growth and progression.

A protocol to co-inject cancer cells and fibroblasts and monitor tumor growth over time is provided. This protocol can be used to understand the molecular basis for the role of fibroblasts as regulators of tumor growth.

研究癌症相关成纤维细胞在肿瘤生长中的作用的小鼠模型

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role ...

Analysis of Lymph Node Volume by Ultra-High-Frequency Ultrasound Imaging in the Braf/Pten Genetically Engineered Mouse Model of Melanoma

Tyr::CreER+,BrafCA/+,Ptenlox/lox&#160;genetically engineered mice (Braf/Pten mice) are widely used as an in vivo model of metastatic melanoma. Once a primary tumor has been induced by tamoxifen treatment, an increase in metastatic burden is observed within 4-6 weeks after induction. This paper shows how Ultra-High-Frequency UltraSound (UHFUS) imaging can be exploited to monitor the increase in metastatic involvement of the inguinal lymph nodes by measuring the increase in their volume.
The UHFUS system is used to scan anesthetized mice with a UHFUS linear probe (22-55 MHz, axial resolution 40 &#181;m). B-mode images from the inguinal lymph nodes (both left and right sides) are acquired in a short-axis view, positioning the animals in dorsal recumbency. Ultrasound records are acquired using a 44 &#181;m step size on a motorized mechanical arm. Afterward, two-dimensional (2D) B-mode acquisitions are imported into the software platform for ultrasound image post-processing, and inguinal lymph nodes are identified and segmented semi-automatically in the acquired cross-sectional 2D images. Finally, a total reconstruction of the three-dimensional (3D) volume is automatically obtained along with the rendering of the lymph node volume, which is also expressed as an absolute measurement.
This non-invasive in vivo technique is very well tolerated and allows the scheduling of multiple imaging sessions on the same experimental animal over 2 weeks. It is, therefore, ideal to assess the impact of pharmacological treatment on metastatic disease.

Melanoma is a very aggressive disease that quickly spreads to other organs. This protocol describes the application of ultra-high-frequency ultrasound imaging, coupled with 3D rendering, to monitor the volume of the inguinal lymph nodes in the Braf/Pten mouse model of metastatic melanoma.

超高频超声成像分析黑色素瘤Braf/Pten基因工程小鼠模型中的淋巴结体积

Tyr::CreER+,BrafCA/+,Ptenlox/lox&#160;genetically engineered mice (Braf/Pten mice) are widely used as an in vivo model of metastatic melanoma. Once a ...

Molecular and Immunologic Techniques in a Genetically Engineered Mouse Model of Gastrointestinal Stromal Tumor

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the KIT receptor. Across tumor types, numerous mouse models have been developed in order to investigate the next generation of cancer therapies. However, in GIST, most in vivo studies use xenograft mouse models which have inherent limitations. Here, we describe an immunocompetent, genetically engineered mouse model of gastrointestinal stromal tumor harboring a KitV558&#916;/+ mutation. In this model, mutant KIT, the oncogene responsible for most GISTs, is driven by its endogenous promoter leading to a GIST which mimics the histological appearance and immune infiltrate seen in human GISTs. Furthermore, this model has been used successfully to investigate both targeted molecular and immune therapies. Here, we describe the breeding and maintenance of a KitV558&#916;/+ mouse colony. Additionally, this paper details the treatment and procurement of GIST, draining mesenteric lymph node, and adjacent cecum in KitV558&#916;/+ mice, as well as sample preparation for molecular and immunologic analyses.

The goal of this manuscript is to describe the KitV558&#916;/+&#160;mouse model and techniques for successful dissection and processing of mouse specimens.

分子和免疫技术在胃肠道间质瘤基因工程小鼠模型中

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the KIT receptor. Across tumor ...

Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features of a unique and convenient model system. As highly proliferative cells are more susceptible to radiation-induced DNA damage, zebrafish embryos are a front-line in vivo model in radiation research. In addition, this model projects the effect of radiation and different drugs within a short time, along with major biological events and associated responses. Several cancer studies have used zebrafish, and this protocol is based on the use of radiation modifiers in the context of radiotherapy and cancer. This method can be readily used to validate the effects of different drugs on irradiated and control (non-irradiated) embryos, thus identifying drugs as radio sensitizing or protective drugs. Although this methodology is used in most drug screening experiments, the details of the experiment and the toxicity assessment with the background of X-ray radiation exposure are limited or only briefly addressed, making it difficult to perform. This protocol addresses this issue and discusses the procedure and toxicity evaluation with a detailed illustration. The procedure describes a simple approach for using zebrafish embryos for radiation studies and radiation-based drug screening with much reliability and reproducibility.

The zebrafish has recently been exploited as a model to validate potential radiation modifiers. The present protocol describes the detailed steps to use zebrafish embryos for radiation-based screening experiments and some observational approaches to evaluate the effect of different treatments and radiation.

斑马鱼幼虫作为评估潜在放射增敏剂或保护剂的模型

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features ...