这项工作得到了自然科学基金 (No。U1532107) 与上海交通大学生物医学工程项目 (No。YG2014MS53)。作者感谢叶剑英李和燕神对 DECT 和 PET/CT 成像方法的有益评价和技术支持。

这项工作是严格按照上海交通大学实验动物保护和使用指南所制定的标准进行的, 并获得瑞金医院实验室动物伦理委员会批准。
1. 胃癌腹膜转移动物模型
<ol><li>将一个适度分化的 SGC-7901 人胃癌细胞系分为 SGC-7901/sFRP1 组和 SGC-7901/vector 组。培养两组细胞分别在 RPMI 1640 补充10% 胎牛血清, 100 单位/毫升链霉素, 和100µg/毫升青霉素在37° c 的湿化气氛与 5% CO2。</li>
	<li>使用4-6 周老的女性裸 balb/c/c 裸鼠与体重25至 30 g. 在动物设施中的特定无病原体条件下放置动物。</li>
	<li>将小鼠随机分为 sFPR1 过度表达组和 sFPR1 空负荷组。 注: 每组有十裸鼠;二十只小鼠随机分为 TGF-β1治疗组和 TGF-β1对照组, 每只动物笼有多达5只裸鼠。</li>
	<li>建立 sFPR1-overexpression 腹膜转移移植模型组, 通过管理150µL (2 x 106细胞/毫升) 通过腹腔 SGC-7901/sFRP1 细胞的悬浮;SGC-7901/vector 单元被管理以建立空加载组。 注意: 使用例计数方法确定单元格的浓度20。</li>
	<li>
建立腹膜转移移植模型组通过管理150µL (2 x 106细胞/毫升) 悬浮 SGC-7901 细胞通过腹腔。管理的目标治疗 TGF-β1抑制剂 SB431542 后两个星期的增长, 经腹腔注射100µL/10 克体重每隔一天对小鼠的治疗组。
	<ol>
<li>对对照组的小鼠进行生理盐水的相同剂量管理。</li>
	</ol>
</li>
	<li>执行 DECT 和 PET/CT 扫描1天前治疗和1天, 7 天, 14 天, 和21天后治疗。</li>
</ol>2. 腹膜转移动物模型的 DECT
注: 在双能量 CT 扫描仪上实现了动物成像实验 (见材料表)。根据前人的研究, 我们建立了相关的 DECT 成像协议。
<ol><li>DECT 成像协议的设置
	<ol>
<li>在映像控制台计算机上, 选择 "协议管理" 图标进入下一个接口, 然后单击 "协议管理" 选项以查看协议管理屏幕。</li>
		<li>在 "用户协议" 界面中, 选择腹部区域进入腹部协议列表。</li>
		<li>单击协议列表中的空白区域, 然后选择 "新建" 按钮以键入新协议的名称: "动物 DECT 扫描"。按键盘上的 "Enter" 键, 然后在弹出窗口中选择 "侦察" 按钮, 单击 "确定" 以设置侦察系列 (第一个系列)。</li>
		<li>选择 "XY" 模式的 "解剖参考点" 和 "头第一仰卧位" 的 ' 病人方向 '。单击 "自动转移" 选项, 然后选择要上载图像系列的工作站位置。在系列描述中命名 "侦察阶段"。</li>
		<li>在 "查看编辑" 屏幕中, 确保相关的扫描参数设置如下: 开始位置 "和" 结束位置 "选项分别设置在" S50 "和" I50 "," KV "在" 100 "," mA "在" 80 "," 90 ° "为" 横向侦察位置 "," 0 ° "为" AP 侦察位置", 和" 侦察员 WW "在" 400/40 "。</li>
		<li>接下来, 创建非增强扫描的第二个系列。点击 "创建新系列", 在弹出窗口中选择 "轴向" 和 "创建后" 图标。</li>
		<li>将系列命名为 "-c 阶段", 并打开 "显示本地化"。在扫描类型接口中, 选择 "螺旋" 扫描类型和 0.5 s 为 "旋转时间", 单击 "粗速度" 选项设置参数 (探测器覆盖范围在40毫米, 螺旋厚度在0.625 毫米, 音高和速度在 0.516:1/20.62, 旋转时间在0.5 秒) 在弹出窗口, 间隔在0.625 毫米, 龙门乳头在 0, SFOV 选择小体, kV 在 100, 点击 "ma" 然后键入600为手动 ma。</li>
		<li>点击 "侦察参数" 图标, 打开 "侦察选项" 弹出窗口。在侦察模式中选择 "加号";单击 "切片" 图标, 在 "ASiR 安装屏幕" 中选择 "ss50 切片 50%" 模式。设置其余参数如下: DFOV 在25厘米, R/L 和一个/P 中心在0厘米, 侦察类型选择 "Stnd", 矩阵大小在512。</li>
		<li>通过重复步骤2.1.8 为增强扫描创建第三个扫描系列, 将其命名为 "+ C QC 阶段" 并打开 "显示定位器";第一组是动脉相系列的设置。</li>
		<li>在 "扫描类型" 界面中, 点击 "(宝石光谱成像)", 并选择 "螺旋" 扫描类型, 选择协议 "GSI-52" 在 ' 腹式预置选择 ' 窗口。根据非增强扫描设置起始位置和结束位置。<ol>
<li>点击 "侦察参数" 图标, 打开 "侦察选项" 弹出窗口。在侦察模式中, 选择 "加号", 然后在 "选项" 中单击 "QC";其余参数与步骤2.1.8 相同。</li>
		</ol>
</li>
		<li>单击 "R2" 图标, 并在 "启用侦察" 选项卡中选择 "是". 选择 "厚度" 在0.625 和类型0.625 为 "间隔"。打开 "侦察选项" 弹出窗口, 在侦察模式选择 "加号";在 "选择" 选项中, 单击 "单声道" 并将凯文设置为70凯文;在 ASiR 设置窗口中, 选择 "GS40 40%" 模式用于 ASiR 安装。其余参数设置为步骤2.1.8。将此步骤命名为 "+ C 70keV 阶段"。</li>
		<li>单击 "R3" 图标, 并在 "启用侦察" 选项卡中选择 "是". 将 "厚度" 设置为 1.25, 类型0.625 为 "间隔"。打开 "侦察选项" 弹出窗口, 在侦察模式中选择 "加号" 和 "IQ 增强";在 "单声道" 选项中, 单击 "单音", 将凯文设置为70凯文, 然后点击 "数据文件";确保 ASiR 安装符合步骤13。其余参数设置为步骤2.1.8。将其命名为 "+ C 单阶段"。</li>
		<li>单击 "添加组" 以创建两个扫描组, 分别表示门户阶段和延迟阶段。确保每个扫描阶段的 "起始位置" 和 "结束位置" 范围一致, 其余参数与动脉阶段相同。在 "准备组" 中键入延迟时间: 第一组 (动脉阶段) 在0秒, 第二组 (门阶段) 在8秒, 第三组 (延迟阶段) 在十六年代。</li>
		<li>在完成所有设置后, 单击 "接受" 选项以保存协议。</li>
	</ol></li>
	<li>DECT 成像过程
	<ol>
<li>在每次扫描前随机从治疗和对照组中选择裸鼠。将选定的动物置于新的笼子中, 并分别标记它们。</li>
		<li>用清水将小鼠快速带入4小时, 但没有食物或被褥。</li>
		<li>在扫描前1小时将实验小鼠从动物实验中心移走, 并确保在扫描开始之前将老鼠置于一个新的温暖环境中。</li>
		<li>麻醉所有实验小鼠腹腔注射2.5% 戊巴比妥钠 (1.0 毫升/千克体重) 前 DECT 扫描成像, 并确认麻醉深度的脚趾捏反射。在麻醉的时候用眼药膏来防止干燥。 注: 在注射药物时, 确保每个裸鼠头部处于较低位置, 从而减少对内脏器官的损伤。注意注射部位和注射深度。将注射器的尖端放在45°角, 放在右/左下腹内, 并确保针的深度是这样, 以避免注射到肠道和其他器官。</li>
		<li>单击 "新患者" 图标, 输入有关鼠标的基本信息, 包括其患者 ID 和名称。在 "用户协议" 中, 单击 "腹部协议" 并选择 "动物 DECT 扫描" 协议进入操作界面。</li>
		<li>一旦引起麻醉, 将每个鼠标移动到一个动物夹具平台的仰卧位, 并修复它的尾巴与磁带, 以确保它不弯曲。用酒精对尾部进行消毒, 随后将造影剂注入尾部静脉。</li>
		<li>移动 CT 扫描床, 使外部定位线激光位于动物的下腹部。当定位完成时, 单击 "重置" 按钮。 注意: 在动物的下腹部放置外部定位线, 确保动物尽可能地位于机器的外部, 便于造影剂的管理进入尾部静脉。</li>
		<li>单击 "确认" 图标, 并按照键盘上按钮的闪烁顺序完成侦察扫描。在完成侦察扫描后, 选择 "下一序列" 图标, 并输入用于非增强扫描的界面。</li>
		<li>在右屏幕上, 设置 "开始位置" 和 "结束位置" 的侦察员视图, 以定义扫描范围。在 "横向侦察" 和 "AP 侦察" 中保持相同的范围, 并覆盖整个动物的身体体积。</li>
		<li>单击 "确认" 图标, 并按照键盘上按钮的闪烁顺序完成侦察扫描。</li>
		<li>通过尾静脉注射每只帕 0.2 mL/100 g 的小鼠。 注: 我们选择手动管理对比剂, 保持注塑速度尽可能稳定。这是最有利于捕捉早期增强肿瘤在成像。</li>
		<li>单击 "下一系列" 以执行增强扫描。根据非增强扫描设置 "开始位置" 和 "结束位置"。单击 "确认" 图标, 并按照键盘上按钮的闪烁顺序完成动态增强扫描、includingarterial 阶段、门户阶段和延迟阶段。 注: 在注射造影剂后立即单击 "确认" 图标开始扫描。这对于增强扫描以确保捕获动脉相的最佳图像至关重要。但是, 单击 "确认" 图标后的一些延迟时间可以确保实验人员从扫描室安全地撤回。</li>
		<li>点击 "结束考试", 在扫描完成后退出扫描界面;图像系列将自动上载到工作站。</li>
		<li>完成扫描后, 将动物放到一个空笼子里, 观察它们, 直到它们恢复知觉。不要让动物无人看管, 直到它恢复了足够的意识来维持胸骨卧床。然后把老鼠转移到一个干净的动物房间。</li>
	</ol></li>
	<li>后 DECT 成像分析
	<ol>
<li>在 DECT workstationinterface 上找到鼠标系列 (请参阅材料表) 并选择 "+ C 单阶段" 系列列表。打开 "加密卷查看器", 并从 "协议管理器" 接口中选择 "一般" 协议。</li>
		<li>单击图像视左上角的 "视图类型" 活动注释, 然后从下拉菜单中选择 "日冕" 方向。</li>
		<li>对于一个图像视区, 单击左上角的 "卷 1" 活动注释, 然后从下拉菜单中选择 "单声道" 卷。同样, 在另一个图像视区中, 选择 "碘 (水)" 卷。单击并按住鼠标左键, 将 "碘 (水)" 视区中的图像拖动到 "单声道", 并检查 "混合视图" 框以获得颜色融合图像。</li>
		<li>单击并从 "图像滚动" 图标的中心拖动以观察图像。将显示阳性结果的图像保存为彩色融合图像。</li>
	</ol></li>
</ol>3. PET/CT 在腹膜转移动物模型中的研究
注: 见所用的 PET/CT 成像仪的材料表。我们根据本文21创建了相关的 PET/CT 成像协议。
<ol><li>
设置微 PET/CT 成像协议
	<ol>
<li>对于全身 CT 扫描, 设置电流在500µA, 电压在80伏, 曝光时间为200毫秒, 240 步为240旋转。对于 x 射线探测器, 选择分辨率在 "低系统放大" 与78毫米轴向成像场和单人床模式。采用 "普通锥梁重构" 方法, 选择 "实时重建" 选项, 使上位机能够与专用的实时重建计算机 (眼镜蛇) 连接, 启动任务。</li>
		<li>对于 PET 的采集, 在 "获取时间" 选项设置 "固定扫描时间" 到 600s (10 分钟)。设置 "研究同位素" 到 F-18 和 "能量水平" 到350-650 凯文。</li>
		<li>为了生成 PET 直方图, 将 "动态帧" 设置为 "黑色", 以便在整个持续时间内将数据作为一个帧来处理, 以实现静态扫描。将直方图类型设置为 "3D", 并选择 "无散射校正" 选项。</li>
		<li>对于 pet 重建, 使用 OSEM3D 算法重建图像, 然后是由 PET/CT workstationsoftware 提供的地图或快速映射22 (请参阅材料表)。</li>
	</ol>
</li>
	<li>
PET/CT 成像前的制备
	<ol>
<li>快速进行4小时 DECT 实验的小鼠, 在成像前30分钟将小鼠转移到新的动物笼中。</li>
		<li>称量小鼠并记录它们的重量。</li>
		<li>按照研究所的安全程序获取和携带含有放射性物质 (RAM) 的包裹。使用防护罩进行18f-葡萄糖 (5 mCi), 并用剂量校验仪测量总的18f-葡萄糖的放射性。</li>
		<li>用生理盐水稀释18F-葡萄糖对小鼠注射液的适当放射性。 注: 18F-葡萄糖的稀释活性应为每只老鼠提供100-200 µCi/100 µL。<ol>
<li>绘制200µL 18F-葡萄糖溶液成1毫升注射器。用剂量校验仪测量整个注射器的放射性, 并记录18F-葡萄糖的准备时间。</li>
		</ol>
</li>
		<li>使用200µL 18f-葡萄糖溶液通过尾部静脉注射路径注入每个鼠标, 并记录18f-葡萄糖注射液时间。注射完所有小鼠后, 立即用剂量校验仪测量注射器的残余放射性, 记录注射完成后测量的时间。</li>
		<li>按以下公式计算每个鼠标的插入的18F-葡萄糖活性: 注射活性 (µCi) = 注射前注射器活性在注射器注入后的活性。</li>
	</ol>
</li>
	<li>
PET/CT 成像过程
	<ol>
<li>将动物放入麻醉诱导室;麻醉小鼠使用吸入3% 异氟醚在氧后完成了18F-葡萄糖注射液。 注意: 遵照所有动物福利准则进行操作;使用加热垫使小鼠保持温暖。用眼药膏预防麻醉时的干燥。</li>
		<li>一旦引起麻醉, 将鼠标移动到微 CT 扫描床上, 同时保持持续的麻醉和变暖。将鼠标的头部放在一个锥面掩模内, 在氧气中连续输送异氟醚 (2%), 流速为2升/分. 将鼠标放在仰卧位, 以确保其姿态与 DECT 扫描中的姿势一致。</li>
		<li>把动物移到宠物/CT 扫描仪的入口处, 单击工具栏查看器中的 "激光" 图标, 并使用触摸板控制界面移动床, 使鼠标的腹部位于 PET 和 CT 场的中心 (视) 扫描过程中。在 "激光对准" 窗口中, 选择 "第一扫描类型" 作为 "CT 扫描", "工作流中包含的 PET 捕获" 作为选择。</li>
		<li>打开 "侦察员视图" 窗口, 并获得一个侦察兵查看 X 射线照相。调整动物床的位置, 使 CT 的中心视野位于鼠标身体的中心。</li>
		<li>选择以前建立的协议 (在步骤3.1 中)。在弹出窗口中输入要映像的鼠标数 (先后), 然后单击 "设置" 选项, 然后输入权重。然后再次单击 "安装" 选项, 然后按照弹出窗口说明完成安装。</li>
		<li>单击 "启动工作流" 图标开始扫描。</li>
		<li>在所有扫描完成后, 评估获得的 CT 和 PET 图像的质量。将数据通过网络传送到后成像分析中进行进一步的研究。 注意: 调整图像的窗口宽度和窗口级别, 以确保正确显示各器官的对比度。检查图像中各器官的分辨率, 确认成像质量。</li>
		<li>将动物从成像仪中取出, 立即安乐颈椎脱位。连续使用下一动物的成像系统。</li>
	</ol>
</li>
	<li>
后 PET/CT 成像分析
	<ol>
<li>打开 pet/ct 工作站软件, 将 ct 和 pet 图像系列数据导入软件中。在 "注册" 窗口中, 单击 "一般分析" 选项, 将 ct 和 pet 图像进行注册, 并在 "查看" 窗口下选择 "天空" 模型, 以显示 ct 和 pet 图像之间的完美匹配。</li>
		<li>在 "感兴趣区域 (ROI) 量化" 窗口中, 通过 co 注册图像提供的参考来识别腹膜结节。</li>
		<li>在 "感兴趣区域 (roi) 量化" 窗口中, 用工具在熔融图像上绘制 roi, 编辑 roi 的大小和形状, 记录 SUV 的max值, 然后输出并保存所选的合并图像。</li>
	</ol>
</li>
</ol>

<ol>
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作者没有利益冲突声明。

动物模型在胃癌的分子机制研究中得到了广泛的应用, 并尝试了各种治疗策略23,24,25。在这项研究中, 我们描述了一个详细的协议, 胃癌腹膜转移裸鼠模型, 使用 DECT 和 PET/CT 图像胃肿瘤实时识别肿瘤细胞增殖, 并监测腹膜转移和胃癌动物模型的治疗干预反应。这种方法可以使参与研究胃癌的分子机制的研究人员, 或实验成像肿瘤, 建立更完整的和精确的计划。此外, 我们描述了 DECT 和 PET/CT 设备的使用, 可以作为发现肿瘤影像学表现与基因调控和靶向治疗的平台。因此, 这种方法可以被科学家用来理解和探索癌症复发和进展的生物学过程。我们已经证明, 非侵入性成像模式可以检测出增加肿瘤的基因过度表达的 DECT 和 PET/CT 阳性结果. 同时, 靶向治疗干预后腹膜转移抑制剂在 PET/CT 上显示负葡萄糖吸收性能。SUVmax 瘤结节呈下降趋势, 延长治疗周期。
在我们的研究中, 我们利用葡萄糖摄取的变化作为评估指标的治疗效果的腹膜转移。已经提出了巨大的努力, 以表明葡萄糖摄取与肿瘤侵袭性相关的26。进展性胃癌, 以浸润深度、淋巴渗透、血管浸润和肿瘤大小为代表, 表现出较高的葡萄糖摄取量27。在定量评价方面, 研究表明, SUVmax与各种恶性肿瘤的增殖呈正相关,15,28。我们的结果表明, 与对照组相比, sFRP1 过度表达积极诱导明显较大的结节, 显著提高和较高的葡萄糖摄取率在腹膜肿瘤, 如在图 1中证明。此外, SUVmax 在靶向治疗组中表现出明显的改变趋势, 对照组无变化。这些结果显示在图 3中, 其葡萄糖摄取量减少, 治疗组的 SUVmax 接近 0.83, 而对照组中的小鼠则不变的 SUVmax 值接近 1.26 (图4).我们的研究结果表明, 非侵入性成像技术, 如 DECT 和 PET/CT, 提供了利用图像技术来评估肿瘤细胞分子水平的信息的可能性, 并证明了结合 DECT 的应用的有效性和 PET/CT 提供一个可行的, 重现性的, 非侵入的成像策略, 以监测肿瘤诱导的基因调控胃癌的研究。由于葡萄糖摄取与肿瘤侵袭性相关26, 利用 PET/CT 成像评估肿瘤浸润和治疗的程度是可行的。
在以往的研究中, 我们发现注射时间和方法会影响 DECT 扫描的成像结果。随着小鼠血液循环的快速更改, 腹膜转移的增加主要发生在动脉期, 如果扫描开始时间明显晚于注射时, 腹膜转移结节可能不会完全显示。在检查后, 裸鼠应放置在一个温暖和清洁的环境中, 让小鼠在 pet/ct 成像前休息12小时, 以避免在 pet/ct 成像中使用过量的残留造影剂对结核的摄取产生影响18F-葡萄糖身体.在葡萄糖的制备和注射过程中应注意注射时间和活动。PET/CT 的最佳活性浓度为 100-200µCi/100µL, 每次注射均为18。浓度过高可能会加重循环系统负担, 导致小鼠死亡, 而浓度过低则会影响肿瘤摄取成像。因此, 确保18F-葡萄糖的配置的效率和准确性至关重要。确保裸鼠的 PET/CT 成像位置与 DECT 成像相一致, 以利于肿瘤图像的匹配。
我们的研究有几个限制。DECT 的有限分辨率可能会导致可见性的负面表现, 因为一些腹膜肿瘤可能表现得不够大。众所周知, PET/CT 的特异性低, 缺乏解剖定位, 化疗药物干预诱导的肿瘤细胞凋亡和坏死可能影响18F-葡萄糖摄取量29,30.此外, 肠道循环正常的生理活动和18F-葡萄糖在输尿管和膀胱中的保留可能有助于假阳性出现在 PET/CT 图像31。裸鼠腹膜转移过程中的模型难以检测和监测, 因此在治疗干预和影像学实验中选择合适的时间尤为重要。因此, 小肿瘤的早期诊断仍然是一个亟待解决的问题。
最后, 我们描述了一种利用 DECT 和 PET/CT 成像技术的方法, 用于准确检测和评价靶向治疗的疗效。我们的结果表明, 非侵入性成像使用所述的协议允许监测和评估腹膜转移进展使用动物模型。该方法的应用将很容易适应临床前研究, 旨在发现胃癌腹膜转移, 这可能是有用的评估疾病诊断或治疗模式。

胃癌 (GC) 仍然是第四最常见的恶性肿瘤, 也是全球癌症死亡率的第二大原因1。虽然胃癌的诊断和治疗的准确性有了很大的提高, 但腹膜转移是胃癌预后和复发的最关键因素, 是术后死亡的决定性指标2。人们普遍认为腹膜传播是一种危及生命的转移方式, 其中, 一旦建立腹膜播散, 疾病就无法控制, 病人的预后较差。因此, 胃癌腹膜转移的检测和疗效评价是临床应用的关键。
胃癌发病率和死亡率的上升促使研究人员识别其分子机制。高表达的基因, 如分泌的卷曲相关蛋白 1 (sFRP1) 可能导致激活的信号通路在早期阶段的胃癌, 促进肿瘤的生长, 增殖, 分化和凋亡的过程中3,4,5,6,7. sFRP1-overexpression 细胞显示 TGFβ的表达增加, 其下游目标, 和 TGFβ介导的急诊室8。以往的研究表明, TGF-β1水平与腹膜转移和胃癌 TNM 分期相关。我们描述了 sFRP1 过度表达和 TGF-β1抑制的癌细胞增殖的变化, 并建立了腹膜转移的动物模型, 以显示肿瘤在基因调控作用下的影像表现。
动物模型的胃癌是必不可少的工具, 研究肿瘤的发展和试验的各种治疗策略, 而不必牺牲动物。动物模型在研究肿瘤和起源细胞的形成机制、确定肿瘤干细胞的存在以及研究各种新的治疗策略方面证明是有用的。因此, 实时无创技术可以准确描述胃肿瘤的发展和治疗的肿瘤反应, 从而确定裸鼠腹膜转移结节的发展, 并监测肿瘤反应的各种实验和治疗干预。
目前, 多探测器 CT (MDCT) 在胃癌 TNM 分期中起着重要作用, 对预测肿瘤切除术后9有一定的参考价值。然而, 经病理证实的胃癌患者的影像学研究主要基于形态学。DECT 成像扩展参数, 以反映功能信息, 提供单色图像, 并可能有助于提高 N 分期准确性胃癌。此外, 这项技术将有助于获得材料分解图像, 这可能有助于区分分化和未分化的胃癌, 并之间转移和非转移淋巴结10.随着 DECT 的引入, CT 的功能成像方面也被添加到临床应用中, 有助于评价疗效和预测患者预后11,12,13。PET/CT 是一种有用的成像技术的检测和分期胃癌, 并能评估复发的肿瘤有效14。肿瘤细胞增殖和血管生成都被认为是必要的, 在开发一个可检测的肿瘤15, 肿瘤结节显示了一个积极的性能与更高的 SUVmax的 PET/CT. 基于对好氧的偏好糖酵解, 18F-葡萄糖的模拟, 已被利用作为一个有希望的示踪剂的诊断恶性肿瘤, 结合 PET/CT16。这种方法依赖于肿瘤组织的快速葡萄糖消耗, 并具有广泛的临床应用, 包括协助检测、分期和评估肿瘤的预后, 以及监测肿瘤对治疗的反应17,18. 由于非侵入性的方法, DECT 和 PET/CT 已被用于诊断恶性肿瘤和评估肿瘤对各种疗法的反应。
我们的小组一直使用这种非侵入性成像方法与 DECT 和 PET/CT 扫描仪, 以检测和监测的过程中的肿瘤生长和转移的活鼠19。我们探讨了 sFRP1-overexpression 诱导的胃癌细胞的影像学表现在体内使用裸鼠, 与 DECT 和 PET/CT, 并描述了 SUVmax值的变化后, TGF-β1抑制剂的靶向治疗, 以确认研究了基因诱导后腹膜肿瘤结节的发展, 并对实验性治疗中肿瘤结节的变化进行了探讨。本文对小鼠胃肿瘤腹膜转移的建模、DECT 和 PET/CT 的检测与监测提供了详细的方法。

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		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Iohexol</td>
			<td style="width:389px;">BEJING BEILU PHARMACEUTICAL CO,LTD</td>
			<td style="width:136px;">NMPN:H20053800</td>
			<td style="width:423px;">non-ionic contrast medium for DECT scan</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">normal saline</td>
			<td style="width:389px;">HUNAN KELUN PHARMACEUTICAL CO,LTD</td>
			<td style="width:136px;">NMPN:H43020455</td>
			<td>placebo of control group</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">BALB/c nude mice&#160;</td>
			<td style="width:389px;">SLAC LABORATORY ANIMAL</td>
			<td style="width:136px;">BALB/cASlac-nu</td>
			<td>animal model</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:31px;width:247px;">SGC-7901&#160; cells</td>
			<td style="width:389px;">Library of typical culture of Chinese academy of sciences</td>
			<td style="width:136px;">TCHu 46</td>
			<td>gastric cancer cell&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">SB431542</td>
			<td style="width:389px;">Selleck</td>
			<td style="width:136px;">No.S1067</td>
			<td>TGF-&#946;1 inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">GE Discovery CT750 HD</td>
			<td style="width:389px;">GE Healthcare</td>
			<td style="width:136px;"></td>
			<td>dual-energy spectral CT scanner&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">AW Volumeshare5</td>
			<td style="width:389px;">GE Healthcare</td>
			<td style="width:136px;"></td>
			<td>dual-energy spectral CT workstation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Siemens Inveon micro-PET/CT</td>
			<td style="width:389px;">Siemens Preclinical Solution</td>
			<td style="width:136px;"></td>
			<td>positron emission tomography/ 
			computed tomography scanner&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Inveon Acquisition Workplace</td>
			<td style="width:389px;">Siemens Preclinical Solution</td>
			<td style="width:136px;"></td>
			<td>PET-CT workstation</td>
		</tr>
	</tbody>
</table>

gene regulation and targeted therapy in gastric cancer peritoneal metastasis: radiological findings from dual energy ct and pet/ct

胃癌在全世界的癌症发病率中仍然是第四, 五年的存活率只有 20%-30%。腹膜转移是胃癌最常见的转移类型, 是预后的决定性因素。预防和控制腹膜转移的发展, 有助于延长胃癌患者的生存期。非侵入性和高效的影像学技术将帮助我们识别腹膜转移的侵袭和转移过程, 并监测肿瘤结节的变化, 以应对治疗。这将使我们能够准确地描述胃癌的发展过程和分子机制。我们最近用双能量 CT (DECT) 和正电子发射断层扫描/计算机断层扫描 (PET/ct) 平台进行了实验, 以检测和监测裸鼠胃肿瘤转移的模型。我们已经表明, 每周连续监测与 DECT 和 PET/CT 可以识别动态变化的腹膜转移。sFRP1-overexpression 在胃癌模型中的作用表现为阳性的放射性表现, 较高的葡萄糖摄取和增强, 而 SUV 的max (标准摄取值) 显示出明显的改变趋势对 TGF-β1抑制剂靶向治疗的反应。在本文中, 我们描述了详细的非侵入性成像程序, 以进行更复杂的研究胃癌腹膜转移使用动物模型, 并提供了代表性的成像结果。利用非侵入性成像技术, 我们应该能够更好地了解肿瘤发生的机制, 监测癌症的生长, 并评估治疗干预对胃癌的影响。

在两周的细胞线注射后, 对裸鼠进行了 DECT 和 PET/CT 扫描。影像学图像在 sFRP1 过度表达组的腹部轮廓外显示皮下转移, 并通过彩色比例图像 (图 1-c) 进行外周增强的转移, 取得了良好的效果。PET/CT 图像描述局部异常葡萄糖摄取转移, 包括在腹膜和皮下转移 (图 1d)。DECT 和 PET/CT 图像上显示的腹膜转移和大皮下转移进一步说明了大体标本和组织学切片 (图 1e-f)。与阳性表达组相比, 在 sFRP1 空载组的腹腔内, DECT 和 PET/CT 图像 (图 2a-b) 没有明显的病灶, 显著的增强, 或高的葡萄糖摄取。虽然大体标本和组织学结果的图像证实了该组的成功植入 (图 2c-d)。
TGF-β1抑制剂和 placebowere 对裸鼠进行两周细胞线注射后的干预治疗, 两周后裸鼠进行 DECT 和 PET CT 扫描。为确定从靶向治疗开始的小鼠腹膜转移结节的形成过程, 进行了随访 DECT 和 PET/CT 扫描。进行非侵入性影像扫描, 以评估 TGF-β1靶向治疗的效果。TGF-β1治疗组的小鼠图像显示, DECT 和 PET/CT (图 3a-b) 的冠状融合图像中明显增强和焦点异常的葡萄糖摄取转移。毛标本只说明腹膜转移灶的8结节 (图 3c), 腹腔扩散分布。定量地,图 3显示了 DECT 的适度外围增强和降低的葡萄糖摄取量, 而 SUVmax接近0.83。另一方面, 对照组正常生理盐水的小鼠在 DECT 和 PET/CT (图 4a-b) 的冠状融合图像中也显示出明显的病灶和局部异常摄取转移。总标本说明了22例腹膜转移瘤 (图 4c), 局部转移结节附着于腹腔。对照组小鼠的肿瘤 SUVmax 值未发生改变 (1.26)。
值得注意的是, 有时肠道会导致葡萄糖轻度摄取和图像中的明亮区域会产生假阳性结果。心脏和膀胱也会聚集大量的葡萄糖, 这可能显示为一个亮点的图像。有必要避免相关的水平图像, 以确定真正的葡萄糖摄取面积的肿瘤。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56526/56526fig1.jpg"> 图 1: 图 sFRP1 过度表达组腹膜转移模型的 DECT、PET/CT 及相应 he 染色.(a-c)在门户阶段的单色图像: (a) 横向单色图像, (b) 横向彩色融合图像, (c) 日冕色标图像;(d) PET/CT 的冠状融合图像 (e) 总标本 (f) 组织学切片。箭头表明腹膜转移结节, 而箭头表明皮下转移。图 1f是肿瘤结节的病理染色结果;图像显示肿瘤细胞的形态和分布;缩放条 = 100 µm。此图已从引用19中进行了修改。<a href="//cloudflare.jove.com/files/ftp_upload/56526/56526fig1large.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/56526/56526fig2.jpg"> 图 2: 图 sFRP1 空负荷组腹膜转移模型的 DECT、PET/CT 及组织学分析.(a) 在门阶段获得的彩色比例融合图像, (b) 融合 PET/CT 图像, (c) 总标本, 和 (d) 组织学部分。腹腔内无可见病灶, 明显异常增强或高葡萄糖摄取。(c) 和 (d) 确认成功植入。箭头在图 2c中指出了由 SGC-7901/vector 细胞系引起的腹膜转移结节。心脏和膀胱在图 2b中显示了明显的葡萄糖升高。图 2d是肿瘤结节的病理染色结果;图像显示肿瘤细胞的形态和分布;缩放条 = 100 µm。此图已从引用19中进行了修改。<a href="//cloudflare.jove.com/files/ftp_upload/56526/56526fig2large.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/56526/56526fig3.jpg"> 图 3: 图 TGF-β1治疗组腹膜转移模型的 DECT 和 PET/CT 及相应的大体标本.(a) DECT 的冠状融合图像 (b) PET/CT 的融合图像. (c) 总标本。心脏和膀胱显示明显的葡萄糖海拔在图 3b。总标本说明8例腹膜转移瘤。箭头指出了与影像学发现相对应的转移性结节。<a href="//cloudflare.jove.com/files/ftp_upload/56526/56526fig3large.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/56526/56526fig4.jpg"> 图 4: 图 TGF-β1对照组腹膜转移模型的 DECT 和 PET/CT 及相应的大体标本.(a) DECT 的冠状融合图像 (b) PET/CT 的熔融图像, 以及 (c) 总标本。总标本说明22例腹膜转移瘤。箭头指出了与影像学发现相对应的转移性结节。<a href="//cloudflare.jove.com/files/ftp_upload/56526/56526fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Supplementary Figure 1" src="/files/ftp_upload/56526/56526supfig1.jpg"> 补充图 1: 样本横向颜色融合图像.<a href="//cloudflare.jove.com/files/ftp_upload/56526/56526supfig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Supplementary Figure 2" src="/files/ftp_upload/56526/56526supfig2.jpg"> 补充图 2: 样品冠状颜色融合图像.<a href="//cloudflare.jove.com/files/ftp_upload/56526/56526supfig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Gene Regulation and Targeted Therapy in Gastric Cancer Peritoneal Metastasis: Radiological Findings from Dual Energy CT and PET/CT at JoVE.com

Gene Regulation and Targeted Therapy in Gastric Cancer Peritoneal Metastasis: Radiological Findings from Dual Energy CT and PET/CT

本协议描述了双能量 ct 和 PET/ct 成像方法在肿瘤成像和疗效评价中的价值。本文阐述了双能量 ct 和 PET/ct 对胃癌腹膜转移的基因调控和靶向治疗的研究方法和结果。

胃癌腹膜转移的基因调控及靶向治疗: 双能量 ct 与 PET/ct 的影像学研究

Gastric cancer remains fourth in cancer incidence worldwide with a five-year survival of only 20%-30%. Peritoneal metastasis is the most frequent type of ...

gene-regulation-targeted-therapy-gastric-cancer-peritoneal-metastasis

Nanyang Technological University

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Medicine

Unit 1

Cancer

SCIENTISTS IN ACTION

10.7K 次观看.  Shanghai Jiao Tong University School of Medicine. 本协议描述了双能量 ct 和 PET/ct 成像方法在肿瘤成像和疗效评价中的价值。本文阐述了双能量 ct 和 PET/ct 对胃癌腹膜转移的基因调控和靶向治疗的研究方法和结果。

视频: Gene Regulation and Targeted Therapy in Gastric Cancer Peritoneal Metastasis: Radiological Findings from Dual Energy CT and PET/CT

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

在免疫缺陷小鼠的建立和传播人类视网膜母细胞瘤肿瘤

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

科研

医学

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

在癌症的生产和检测活性氧（ROS）的

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

Quantification of Atherosclerotic Plaque Activity and Vascular Inflammation using [18-F] Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography (FDG-PET/CT)

Conventional non-invasive imaging modalities of atherosclerosis such as coronary artery calcium (CAC)1 and carotid intimal medial thickness (C-IMT)2 provide information about the burden of disease. However, despite multiple validation studies of CAC3-5, and C-IMT2,6, these modalities do not accurately assess plaque characteristics7,8, and the composition and inflammatory state of the plaque determine its stability and, therefore, the risk of clinical events9-13.
[18F]-2-fluoro-2-deoxy-D-glucose (FDG) imaging using positron-emission tomography (PET)/computed tomography (CT) has been extensively studied in oncologic metabolism14,15. Studies using animal models and immunohistochemistry in humans show that FDG-PET/CT is exquisitely sensitive for detecting macrophage activity16, an important source of cellular inflammation in vessel walls. More recently, we17,18 and others have shown that FDG-PET/CT enables highly precise, novel measurements of inflammatory activity of activity of atherosclerotic plaques in large and medium-sized arteries9,16,19,20. FDG-PET/CT studies have many advantages over other imaging modalities: 1) high contrast resolution; 2) quantification of plaque volume and metabolic activity allowing for multi-modal atherosclerotic plaque quantification; 3) dynamic, real-time, in vivo imaging; 4) minimal operator dependence. Finally, vascular inflammation detected by FDG-PET/CT has been shown to predict cardiovascular (CV) events independent of traditional risk factors21,22 and is also highly associated with overall burden of atherosclerosis23. Plaque activity by FDG-PET/CT is modulated by known beneficial CV interventions such as short term (12 week) statin therapy24 as well as longer term therapeutic lifestyle changes (16 months)25.
The current methodology for quantification of FDG uptake in atherosclerotic plaque involves measurement of the standardized uptake value (SUV) of an artery of interest and of the venous blood pool in order to calculate a target to background ratio (TBR), which is calculated by dividing the arterial SUV by the venous blood pool SUV. This method has shown to represent a stable, reproducible phenotype over time, has a high sensitivity for detection of vascular inflammation, and also has high inter-and intra-reader reliability26. Here we present our methodology for patient preparation, image acquisition, and quantification of atherosclerotic plaque activity and vascular inflammation using SUV, TBR, and a global parameter called the metabolic volumetric product (MVP). These approaches may be applied to assess vascular inflammation in various study samples of interest in a consistent fashion as we have shown in several prior publications.9,20,27,28

Quantification of Atherosclerotic Plaque Activity and Vascular Inflammation using [18-F] Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography FDG-PET/CT

There is great need to identify atherosclerosis non-invasively, and here we demonstrate how FDG-PET/CT can be used to detect and quantify atherosclerotic plaque activity and vascular inflammation.

动脉粥样硬化斑块的活动和血管炎症的定量使用[18 F]，氟正电子发射断层扫描/电脑断层扫描（FDG-PET/CT）

Conventional non-invasive imaging modalities of atherosclerosis such as coronary artery calcium (CAC)1 and carotid intimal medial thickness (C-IMT)2 ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

利用磁共振成像，容积电脑断层扫描及超音波的裸鼠乳腺癌骨转移大鼠模型血管生成的多模态成像

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

一种新的手术方法气管内注射生物活性剂在胎鼠模型

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

人类前列腺癌转移的原位小鼠模型

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

胆管结扎小鼠:感应炎性肝损伤和纤维化的阻塞性胆汁淤积

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

在体内和体外途径研究银河系结构现货卵巢癌转移定植腹膜脂肪

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

腹膜内卵巢癌转移的定量

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

通过重组腺相关病毒载体注射立体的α-突触核蛋白基于大鼠模型为帕金森氏病的发展

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...