Name: murine model for non-invasive imaging to detect and monitor ovarian cancer recurrence
Uploaded: 2014-11-02T14:00:00.000+00:00
Duration: 8 min 55 s
Description: Watch this Scientific Journal Video about Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence at JoVE.com

这项研究是由美国国立卫生研究院资助RO1CA118678和RO1CA127913支持，由金沙家庭基金会，以及发现治愈方案。

耶鲁大学实验动物管理和使用委员会批准的所有的体内工作。与患者的同意下进行，并批准了耶鲁大学医学院的人类调查委员会的样品采集。 1.准备人卵巢癌细胞<ol><li>之前制备的细胞以确保所需的下述的子宫内注入所有材料都准备好，易于使用。一旦注入细胞悬液，因为它已准备就绪。 <ol><li>生长在培养荧光标记的癌细胞。我们使用一个F2代的人CD44 + / + MyD88的上皮性卵巢癌的细胞，这是我们以前表明窝藏干性性能如致瘤性，化学抗性及对于肿瘤修14-19高容量的。生成这些细胞稳定表达mCherry荧光（F2-mCherry， 图1），使用慢病毒从聚乙烯亚胺产生的（PEI）协议20,21。 </li><li>于注射的当天，通过胰蛋白酶消化收获细胞并用磷酸盐缓冲盐水（PBS）洗一次。 </li><li>确定细胞计数和悬浮细胞以3×10 6细胞/50μl的适当的生长培养基。对卵巢癌细胞，我们使用的RPMI 160有10％FBS。使细胞保持在培养器，直到准备注入，但不保持时间超过15分钟。这是足以让一只老鼠注射。 </li></ol></li></ol>人卵巢癌细胞2.在无胸腺裸鼠宫内​​注射液注意，下面的步骤需要来自第二人的帮助。此外，由于这是存活手术中，使用无菌的手术器械。这生存术过程大约需要10到15分钟。 <ol><li>麻醉，用2％异氟醚的鼠标。 7〜8周龄的无胸腺裸小鼠中，通常使用。 </li><li>确认该动物是COMPLET由俭用手指或镊子垫脚的伊利麻醉。动物应该是完全无反应开始前，手术的痛苦。 </li><li>将麻醉小鼠腹侧的无菌纱布垫与头离调查。 </li><li>用棉花尖端涂药，以防止干燥时在麻醉下应用软膏的眼睛。 </li><li>立即插入头连接到一个异氟烷蒸发器（ 图2A），一鼻锥系统。这也提供了在整个手术过程中麻醉。 </li><li>消毒的左（或右）的腹部用酒精垫（ 图2B），接着加入碘侧然后将无菌外科手术布在外科手术部位。 </li><li>用无菌手术剪和镊子进行1-2厘米的皮肤切口上的鼠标（ 图2C）的左下象限。 </li><li>解除肌肉和做一个切口，以达到腹膜。 </li> &lt;利&gt;解剖的斜肌以暴露腹腔。 </li><li>找到并确认左宫角（ 图2D）。 </li><li>使用无菌止血钳，夹紧角（ 图2E）的两个前和后侧面。放置右输卵管下面的前夹紧和后夹紧权利子宫颈以上。 </li><li>有第二人注入细胞悬液。将装有细胞的针，在45°角沿垂直于喇叭（ 图2F）。非常缓慢地注入50微升细胞悬浮液放入子宫角的内腔中。 </li><li>取出针和释放前夹紧。 </li><li>松开后夹紧和替换子宫角在腹腔内。 </li><li>使用合成的可吸收缝合关闭腹膜和使用组织粘合剂闭合皮肤。 </li><li>从鼻锥取下鼠标放回笼子。如有必要，修改的床上用品，以确保CLEA手术在n笼。确保所有的动物都清醒和主动离开前无人值守。 </li><li>提供布洛芬在饮用水中的第48小时的手术后的0.11毫克/毫升。随后，换上普通的饮用水。 <ol><li>注意:如果无胸腺裸鼠的使用和皮肤，使用组织粘合剂代替缝合关闭，这是没有必要的手术后，分离小鼠。 SCID小鼠中更积极的，并且可以具有被分离。 </li></ol></li><li>密切监测切口可能的感染部位，由于开放性伤口。 </li></ol> 3.检测早期腹膜内卵巢癌的活体内成像确定腹腔IP疾病的体内成像存在了现场。多模态动物轮作系统（MARS）用于本协议。 <ol><li>从MI软件中访问MARS捕获软件。该模块提供在火星协调控制拍摄设置，同时充分利用了成像系统的捕获和分析能力。使用前，开发的旋转协议在步骤3.4单视图影像优化模式每个单独的拍摄设置。 </li><li>输入预定的捕捉值。对于荧光采集，使用10秒曝光，2×2像素合并，光圈2.8，视场120毫米，例如:550纳米，EM:600纳米过滤器捕获设置。对于X射线拍摄，使用10秒曝光，2×2像素合并，光圈2.8，视场120毫米，35 kVp的0.4mm的铝过滤器捕获设置。 </li><li>保存参数步骤3.2作为独立的会话文件。 </li><li>由来自系统接口获取窗口选择创建/编辑协议按钮创建一个旋转序列协议。 </li><li>选择之前保存的荧光会议文件，并保存为第一步。接下来，选择相应的透视会话文件，并保存为第二步。 </li><li>与第一步中选择， 使用 Set 旋转系列从之前图像捕捉弹出菜单中设置所需的起始角度，范围和增量，以确保功能的无缝可视化。特定的值包括为-180°起始角，且具有375度，和15度的增量。 </li><li>保存该协议，然后单击完成按钮初始化MARS和地图对应于每个角度增量的步进电机的位置。 </li><li>如下所述对齐的腹侧，背侧和横向视图定位在动物支持具有特定度的旋转的具体动物要使用软件协议执行最后的校准。 </li><li>麻醉用医用空气和2％异氟醚的混合物以2升/分钟的流速鼠标。 </li><li>从采集菜单中选择预览按钮，弹出肩选项卡启动校准。使用反射光模式，而不是多波长或X-RAŸ方式来加快这一进程。 </li><li>选择加载鼠标选项，并继续通过一系列的定位菜单。 </li><li>确保可折叠鼻锥的管很端是在鼻锥凹部，然后将鼠标在俯卧的取向，其在鼻锥头。 </li><li>开始校准在朝下的动物的腹侧面的0度位置。使用旋转系统上的两个旋钮来定位动物，使其居中显示在预览窗口中。 </li><li>重复上述过程，提示-180， - 90，90，和180度的位置，然后单击完成 。 </li><li>通过点击主窗口获取的执行协议按钮执行以前保存的协议。在协议的执行，观察状态窗口为当前捕获，暴露时间，以及整体协议的步骤提供实时状态更新。 </li><li>个人组装成图像使用电影格式旋转的软件。使用显示控制来调整亮度/对比度，透明度，以及设置显示颜色的荧光信号。 </li><li>在导出的介绍* .AVI文件格式的图像的最终序列。 </li></ol> 4.政府1日和第二轮化疗<ol><li>以下的ip肿瘤检测由MARS，使用标准的二维定位确定的感兴趣区域（ROI）的初始肿瘤负荷区域。这是，如第5节如下所述。一旦初始的投资回报率是确定的，施用4剂量的12毫克/公斤的紫杉醇的ip每三天装在成像系统中的透明动物托盘进行。紫杉醇是从Hospira的公司获得一个现成的配方去与cremaphor车辆。我们以前表明，此治疗方案具有抗异种移植模型显著的抗肿瘤活性与载体对照相比，诱导彻底根除可检测吨umor负担20。然而，由于该疾病的性质和ip肿瘤复发几天治疗结束后20。 </li><li>监测对治疗的反应通过使用标准的二维定位成像每隔3天。 </li><li>紫杉醇的第四给药后，将小鼠归类为具有完全应答率（ROI小于2000），部分应答（减少来自步骤4.1，疾病稳定所得到的初始投资回报率高达35％（最多50上升，从最初的投资回报率％ ），或进展与基于所观察到的荧光信号处理（增加从最初的投资回报率超过50％）。 </li><li>对于小鼠完全缓解，经影像学每​​3天监测复发。一旦复发观察（ROI =初始投资回报率在4.1步），再启动在4.1中描述的第二轮紫杉醇。 </li><li>小鼠局部疾病，疾病稳定，或那些与治疗的进展， 第 4 次剂量后3天重新开始第二轮紫杉醇紫杉醇。同样，图像这些每三天。 </li></ol> 5.监测对治疗的反应使用标准的二维定位<ol><li>麻醉小鼠用医用空气和2％异氟醚的混合物以2升/分钟的流速进行成像。 </li><li>将麻醉的小鼠中个体nosecones的头在成像室（ 图3）内的透明动物托盘。 </li><li>输入为荧光预定的曝光参数（10秒曝光，将2×2像素合并中，f-停止2.8，视场角120毫米，例如:550纳米，青霉:600纳米）和X-射线（20秒曝光，将2×2像素合并，光圈2.8，视场120毫米，35 kVp的0.4mm的铝过滤器）。 </li><li>点击暴露在连续拍摄两幅图像。 </li><li>目视检查和利益分析的区域中打开布鲁克MI软件的图像。 </li></ol> 6.图像叠加和分析为了给博进行有效的比较的时间过程的图像序列处理的所有前景和背景的图像类似的第视觉和定量分析。 <ol><li>在软件中打开两个前景图象（ 即荧光）和背景图像，并选择在顶部（ 图4A）从窗口菜单中瓷砖功能。 </li><li>处理的前景图像;打开顶部栏（ 图4A，红色箭头）的图像显示选项卡，设置最小/最大值为260和5000，分别为（ 图4A，蓝色箭头） 注:图片显示范围不同于研究根据图像中捕捉荧光信号强度来研究。图片应该对比与最小值/最大值，将显示肿块，但没有显示出从动物的自发荧光背景。 </li><li>下次打开自动ROI的标签从导航面板左侧（ 图4B，红色箭头），然后从菜单中选择新建投资回报率设置（ 图4（c），红rrow）。调整设置以使用阈值算法在630级灰度并取消限制规模最大，以确保公司记录所有潜在的投资回报率（ 图4C）。 注:阈值设置可能会有所不同，从研究，研究根据图像中捕捉荧光信号强度。图像阈值化，以定量从肿瘤块信号尚未从动物的自体荧光背景测量信号。 </li><li>从形象展示的窗口中选择屏蔽功能。现在仅对应于具有等于的值或超过630级灰度的阈值的像素的区域将被显示。 </li><li>要查看的定量结果中选择顶部菜单栏和选择显示选项卡（ 图4D，红色箭头）的分析选项卡。 </li><li>从显示菜单中选择序列号，求和，平均值和地区的复选框，然后单击确定。 </li><li>保存图像，这样的值可以是前移植沿着一旦所有荧光图像进行处理。 </li><li>其次，突出透视图像，再从顶部的菜单栏打开的图像显示。设置为最小值，最大值和伽玛值赋予鼠标骨架的最佳视觉表现。 注意:由于没有定量的是，在X射线图像的时间序列中的每个单独的X射线图像可以被处理执行（ 即最小值，最大值和灰度值），以产生数据集的最好看的表示。 </li><li>再次与透视图像高亮选择覆盖复选框，并从下拉菜单（ 图4E）选择相应的荧光图像的文件名 ​​。 </li><li>要提取叠加输出，然后从导航菜单（ 如图4B所示，蓝色箭头）左侧的注释标签。选择适当的选项卡添加任何额外的数据或文本最后添加的力度比例尺这样的荧光强度数据可以快速地从IM确定年龄。 </li><li>一旦标注完成突出显示所有批注页面上显示的对象，并从编辑菜单中选择复制在上面粘贴的叠加图像数据转换成相应的文件（ 例如 Word，PowerPoint和/或Photoshop）。 </li><li>重复步骤6.1- 6.11的序列中所有对荧光和X射线图像。确保安全的所有图像关闭之前。 </li><li>对于最终的定量打开所有的荧光图像的同时，并沿顶部的窗口菜单中选择平铺。 </li><li>下一步选择导出所有打开的文档来查看数据。 注:在没有Excel程序中，取消选择该选项卡。 MI将生成一个制表符分隔的文本可以被转移到一个适当的计算机进行进一步的分析（ 即 .txt）文件。 </li></ol>

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我们描述了一个协议来建立模仿观察患者的临床资料的知识产权人卵巢癌动物模型。此外，我们描述了使用该地址2D成像的灵敏度限制动物的旋转的装置。两者合计，这些技术可以作为平台来发现新的化合物，可以靶向化疗耐药卵巢癌复发。此外，这样的模型可以用来了解癌症复发和进展的生物学特性。 由于它的腹膜后位置，早期的ip卵巢癌异种移植物是几乎不可能通过物理检查鼠标来检测。在大多数情况下，一旦发病可触及的肿瘤负荷是已经显著，因此限制了治疗效果的评价。利用荧光标记的细胞可以让我们实时评估，从而确定最佳时间，开始吨设立知识产权肿瘤reatment。用类似的方法，荧光标记的异种移植物允许监测治疗反应。应当指出超时但是那的ip肿瘤更深超过1厘米通常检测不到的，不论记者系统。 使用人卵巢癌干细胞14,15,17,22的产生模仿的患者中观察到的临床信息异种移植物。作为一个主要的疾病，该模型是响应于紫杉醇但停止治疗最终导致化疗耐药复发性疾病。通过子宫角在协议部分指定的密度引入的细胞通常会导致有一些腹膜种植，因此模仿早期疾病10天内卵巢肿瘤。使用荧光标记的细胞使我们能够评估在实时并因此确定的最佳时间开始治疗建立的ip肿瘤。用类似的方法，荧光标记的异种移植物允许监测ö˚F对治疗的反应。如果其它类型的癌症细胞系的使用，卵巢或其他，这是可能的，这种信息可能不被观察到。当SKOV3被用于例如，已经报道，在初始的ip肿瘤已经抗性23。然而，如果标记报告如荧光和ip，疾病可以遵循​​在实时性。 如果其他荧光报告的情况下，与对照（无肿瘤）的动物进行初始成像是非常重要的。这将允许成像协议的优化，以实现对信号比率的最佳背景。根据我们的经验，裸鼠体内通常有较高的背景下，当使用GFP采集设置成像。 重要的是，细胞注入子宫内都在单个悬架，以避免在子宫建立肿瘤。还有一点很重要，以避免刮伤子宫上皮细胞层，这也方便了癌细胞的植入在子宫s，从而产生一个帧内子宫肿瘤，而不是一个IP疾病。此外，在数据分析中，它设置伽玛值为1。这确保了图像的强度为线性，并允许图像之间的比较是重要的。 在采集MARS的图像，重要的是确保可折叠鼻锥的管很端是在鼻锥凹部是很重要的。鼻锥充当联系点鼠标，因此，需要用于获得精确校准的角度。对于更长的成像方案（ 即长于1小时），注射100微升无菌生理盐水皮下注射，以帮助防止脱水。应保持动物的体温用暖空气通过该系统流在约37℃。 MARS的系统的一个限制是，只有一只动物可以同时进行成像以每只动物约1小时，总的运行时间。 总之，我们描述了ESTablishment的动物模型，该模型密切模仿卵巢癌，原发性和复发性疾病。该模型可用于评价新的诊断或治疗方法的功效。

动物模型是在生命科学研究不可或缺的工具。在癌症特别是，从动物研究获得的数据提供给发起的新的诊断或治疗应用在人类1-3的测试所需的必要信息。动物模型实体癌是古典建立皮下，因为它提供了一种简单的方法来测量肿瘤负荷和评估治疗效果，而不必牺牲动物。的确，腹膜内（IP）的模型要求的动物被处死，以检测和测量肿瘤的生长的任何变化。然而，对于的ip癌症如卵巢癌，正交各向异性模型提供研究疾病在其适当的环境4-6的优点。对于这样的模型是在抗肿瘤活性的评价使用的，非侵入性的成像方法需要开发允许在活体小鼠的ip肿瘤负荷的定量。 在一个主要挑战使用IP动物模型是精确通过体检量化肿瘤负荷的难度。 IP肿瘤的精确定量通常需要被牺牲解剖小鼠。这种方法需要使用大量的动物，这会在不同的时间点被牺牲掉。除了成本，它引入了高数据变异由于每只动物中固有的变化。非侵入性的体内光学成像提供了更合适的方法来监测活小鼠中的ip肿瘤负荷。 几个非侵入性的成像方法目前被用于临床前研究的肿瘤生长和治疗反应的监测。这些包括计算机断层扫描（CT），超声（US），磁共振成像（MRI），正电子发射断层扫描（PET）和光学成像，如荧光和生物发光7-12。 CT是结合X射线和COMPUT传输成像过程呃技术。它产生的高能量光子，穿过体具有不同速度的检测光束的横截面图像。美国是一种类型的反射图像，该发送高频率声音，以被反射的不同速度取决于组织的密度和由计算机识别，以产生视觉图像体内产生的声波。 MRI和PET是使用磁性能和核的颗粒，分别产生图象发射的成像模态。核磁共振产生强大的磁场诱导细胞产生自己的无线电频率，这被用来创建一个图像，而PET需要一个敏感照相机来检测给药标记的2-fluorodeoxy-D-葡萄糖7,9,11的放射性。最后，光学成像是基于生物发光或荧光报告或探头9,12的发射光的检测。 在这份报告中，我们描述了使用荧光，它提供几个优于其它类型的成像模态的。用荧光成像，细胞可以是遗传改造以表达荧光蛋白不断而不需要另外的衬底或结扎基于探针，这是必要的用于生物发光和磁共振成像，分别的。荧光记者通常还表示较亮的信号从而允许使用较不敏感的检测方法8,12的。此外，与荧光成像，它能够检测肿瘤大于1厘米，这是不可能实现的CT 7-9小。最后，相对于生物发光，荧光信号并不需要有氧环境，因此该信号不限于缺氧环境中，这通常是在大的肿瘤13的芯存在。 然而，正如任何其他的技术，基于荧光的成像方法有其缺点。其中之一是町无力网元产生的低能光子穿透到足够的深度。因此，为了最小化的扩散组织的光子的动物应以不同的角度进行成像的量。我们描述了一个协议来建立IP卵巢癌裸鼠移植瘤和肿瘤的IP监控，通过旋转提供整个动物成像的方法。转子角鼠标特定的和可重复的位置降低，往往在光源和检测器之间发生的组织的干扰。这优化了较小的肿瘤可能被遗漏的可视化。

<table><tbody><tr><td>RPMI 1640 media</td><td>GIBCO, by Life Technologies</td><td>23400-021</td><td></td></tr><tr><td>fetal bovine serum</td><td>Gemini Bioproducts</td><td>100-106</td><td></td></tr><tr><td>T75 cell culture flasks</td><td>Corning</td><td>430641</td><td></td></tr><tr><td>PBS</td><td>Life Technologies</td><td>10010-023</td><td></td></tr><tr><td>Trypsin</td><td>GIBCO, by Life Technologies</td><td>25300-054</td><td></td></tr><tr><td>Isoflurane</td><td>Butler Schein</td><td>NDC 11695-6776-1</td><td></td></tr><tr><td>Alcohol pads</td><td>Fischer Scientific</td><td>06-669-62</td><td></td></tr><tr><td>1 ml syringe</td><td>Becton Dickinson</td><td>309602</td><td></td></tr><tr><td>25 gauge needle</td><td>Becton Dickinson</td><td>305122</td><td></td></tr><tr><td>synthetic absorbable suture</td><td>Covidien</td><td>SL-636</td><td></td></tr><tr><td>tissue adhesive</td><td>Vetbond</td><td>1469SB</td><td></td></tr><tr><td>surgical scissors</td><td>VWR</td><td>82027-584</td><td></td></tr><tr><td>surgical forceps</td><td>VWR</td><td>82027-386</td><td></td></tr><tr><td>hemostat</td><td>VWR</td><td>82027-422</td><td></td></tr><tr><td>Paclitaxel</td><td>Hospira, Inc.</td><td>NDC 61703-345-50</td><td></td></tr><tr><td>Ibuprofen</td><td>Walgreens</td><td></td><td>Children&#039;s Ibuprofen 100 (100 mg/5 ml)</td></tr><tr><td>Puralube Vet ointment</td><td>Pharmaderm</td><td></td><td></td></tr><tr><td>In vivo MS FX PRO</td><td>Bruker Corporation</td><td></td><td></td></tr><tr><td>MI software</td><td>Bruker Corporation</td><td></td><td></td></tr><tr><td>athymic nude mice</td><td>Harlan</td><td></td><td></td></tr></tbody></table>

murine model for non-invasive imaging to detect and monitor ovarian cancer recurrence

上皮性卵巢癌是美国最致命的妇科恶性肿瘤。虽然患者初步护理，包括减瘤手术和化疗联合组成的铂类和紫杉类化合物的现行标准做出反应，患者近90％在几年内复发。在这些患者中化疗耐药性疾病的发展限制了目前可用的化疗药物的效果，因此有助于高死亡率。发现新的治疗方案，能针对复发性疾病，适当的动物模型，密切模仿患者的复发性卵巢癌的临床资料是必需的。监测腹腔（IP）的疾病所面临的挑战限制了IP模式，因此大多数移植建立皮下。我们已经开发了一种灵敏的光学成像平台，允许检测和ip肿瘤块的解剖位置。该平台包括的U本身光学记者，从可见光范围到近红外线，从而在与2维X射线共登记组合可提供的分子信号解剖位置延伸。检测是通过使用旋转系统，用于驱动所述动物到多个角位置为360度成像，允许肿瘤不在单一方向可见的标识显著改善。该平台提供了一个独特的模型，以非侵入性地监测肿瘤的生长和评估的用于预防或治疗的复发性卵巢癌的新治疗方法的功效。

非侵入性实时成像允许的IP肿瘤进展来自同一小鼠的监控，通过时间。子宫内注射使用所述协议mCherry-标记F2的卵巢癌细胞中的32天产生明显的ip肿瘤在第5天与癌病（ 图5）。图6示出了所获得的图像与癌病的相关性牺牲小鼠后观察。 使用MARS成像允许如果图象只从一个单一的平面获取的，将被遗漏的ip肿瘤的检测。 图7（上图）表明，即使在对照小鼠与显著肿瘤负荷，肿瘤大小可低估取决于角图像是从。当动物被归类为一个完整的响应，以确保肿瘤块不遗漏或低估的能力，甚至在第一轮化学疗法的结论更重要呃局部响应，或者非应答（ 图7中图）。同样，维持治疗期间，它是最重要的，以检测非常小的复发肿瘤正确指定天复发，其限定无进展的时间间隔（ 图7中图）。 肿瘤负荷定量评估是通过动物旋转优化。动物的旋转角度，将最小化深度的激发和发射的光行进通过将允许从荧光，这表明肿瘤细胞的数量，因而肿瘤负荷的光子的最精确的捕获。因此，定量进行了优化，这取决于其在所述旋转顺序中的位置，在动物的特定的肿瘤。 在介绍的旋转数据集图像，分配图像的最小/最大图像对比度，将有效地显示所有肿块范围，同时允许VISU是很重要的<html>人划定个别肿块的。对于本研究中，理想的对比度范围被发现是最小的50计数和一个最大的1200计数。这些值可以看作是一个指引等的研究，但是，最佳的对比度范围将从研究不同来研究这取决于肿瘤负荷水平，在细胞中的荧光肽的表达水平，成像系统的配置，并拍摄设置。 <img alt="图1" fo:content-width="6in" src="/files/ftp_upload/51815/51815fig1highres.jpg" width="600" /> 图1:F2卵巢癌细胞稳定表达mCherry荧光。 （ 一 ）第一阶段的图像; （B）的荧光图像; A和B（C）覆盖需要注意的是细胞100％表达荧光记者。 s.jpg"宽度="600"/&gt; 图2:宫内注射卵巢癌细胞。 （ 一 ）麻醉持续通过鼻锥管理; （BE）皮肤切开定位和夹紧子宫角; （F）中的癌细胞缓慢注入子宫腔内。 <img alt="图3" fo:content-width="6in" src="/files/ftp_upload/51815/51815fig3highres.jpg" width="600" /> 图3:（A）2D成像与控制和闭路通风的成像室中的透明动物托盘的温度（B，C）盘配有鼻锥能够提供麻醉和可容纳5只小鼠。 <img alt="图4" fo:content-width="6in" src="/files/ftp_upload/51815/51815fig4highres.jpg" width="600" /> &lt;强&gt;图4:从分析软件中抽取代表窗板，以协助数据分析。请参阅文本的解释。 <img alt="图5" fo:content-width="6in" src="/files/ftp_upload/51815/51815fig5highres.jpg" width="600" /> 图5:检测mCherry荧光共定位与X射线在纵向成像序列。三小鼠肿瘤IP遵循通过时间来评估IP肿瘤进展。需要注意的是肿瘤进展可能会发生变化。图3是小鼠肿瘤的进展速度不同，因此，通过随时间变化的肿瘤负荷的代表图像。 <img alt="图6" fo:content-width="6in" src="/files/ftp_upload/51815/51815fig6highres.jpg" width="600" /> 图6:IP肿瘤负荷（上图）和荧光/透视叠加IMA的相关性通用电气（下图）。约32天后F2-mCherry卵巢癌细胞的注射，2D成像被执行并且小鼠处死关联实际肿瘤负荷与所获取的图像。红色箭头指向肿瘤负荷。 <img alt="图7" fo:content-width="6in" src="/files/ftp_upload/51815/51815fig7highres.jpg" width="600" /> 图7:旋转的数据集，允许多角度的成像与检测肿瘤的控制（上图），紫杉醇治疗（中图）和复发性的小鼠（下图）。图中显示，因为它是使用MARS系统旋转每个面板的单个小鼠的图像。需要注意的是，即使在对照小鼠与显著肿瘤负荷，肿瘤大小，可以根据不同的图像，从拍摄的角度低估。

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Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

小鼠模型的非侵入性成像检测和监测卵巢癌复发

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

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12.2K 次观看.  Yale University School of Medicine. We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer. 

视频: Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is important to control peritoneal dissemination. However, it is still unclear how tumor cells detach from primary lesions and attach to the mesothelium. The establishment of an appropriate animal model is needed to gain an understanding of the mechanism of peritoneal dissemination in vivo. In the current study, we introduce the process from the local injection of EOC cells into the murine ovarian surface to the development of metastasis, including the peritoneum and distant organs. Female nude mice (BALB/c nu/nu) at 8 weeks of age were used. Under a microscopic field of view, EOC cells (1 x 105 cells/&#181;l of medium-extracellular matrix (ECM)-based hydrogel/unilateral ovary/mouse) were injected into murine ovaries through a retroperitoneal approach from the dorsal flank. This proposed method is a less invasive procedure for the mouse and minimizes damage to the ovary. Here, we describe the methodological steps in the development of the original and metastatic tumor formation of EOC.

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

To clarify the multistep process of peritoneal dissemination of ovarian carcinoma, the current study presents a murine experimental model of original tumor development and peritoneal metastasis via orthotopic inoculation with these tumor cells.

原发肿瘤发展小鼠实验模型和腹膜转移<em&gt;通过</em&gt;原位接种卵巢癌细胞

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is ...

科研

癌症研究

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all gynecologic malignancies among US women 1,2,3. Serous tumors are the most widespread forms of ovarian cancer and 4,5 the Tg-MISIIR-TAg transgenic represents the only mouse model that spontaneously develops this type of tumors. Tg-MISIIR-TAg mice express SV40 transforming region under control of the Mullerian Inhibitory Substance type II Receptor (MISIIR) gene promoter 6. Additional transgenic lines have been identified that express the SV40 TAg transgene, but do not develop ovarian tumors. Non-tumor prone mice exhibit typical lifespan for C57Bl/6 mice and are fertile. These mice can be used as syngeneic allograft recipients for tumor cells isolated from Tg-MISIIR-TAg-DR26 mice.
Objective: Although tumor imaging is possible 7, early detection of deep tumors is challenging in small living animals. To enable preclinical studies in an immunologically intact animal model for serous ovarian cancer, we describe a syngeneic mouse model for this type of ovarian cancer that permits in vivo imaging, studies of the tumor microenvironment and tumor immune responses.
Methods: We first derived a TAg+ mouse cancer cell line (MOV1) from a spontaneous ovarian tumor harvested in a 26 week-old DR26 Tg-MISIIR-TAg female. Then, we stably transduced MOV1 cells with TurboFP635 Lentivirus mammalian vector that encodes Katushka, a far-red mutant of the red fluorescent protein from sea anemone Entacmaea quadricolor with excitation/emission maxima at 588/635 nm 8,9,10. We orthotopically implanted MOV1Kat in the ovary 11,12,13,14 of non-tumor prone Tg-MISIIR-TAg female mice. Tumor progression was followed by in vivo optical imaging and tumor microenvironment was analyzed by immunohistochemistry.
Results: Orthotopically implanted MOV1Kat cells developed serous ovarian tumors. MOV1Kat tumors could be visualized by in vivo imaging up to three weeks after implantation (fig. 1) and were infiltrated with leukocytes, as observed in human ovarian cancers 15 (fig. 2).
Conclusions: We describe an orthotopic model of ovarian cancer suitable for in vivo imaging of early tumors due to the high pH-stability and photostability of Katushka in deep tissues. We propose the use of this novel syngeneic model of serous ovarian cancer for in vivo imaging studies and monitoring of tumor immune responses and immunotherapies.

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

To study in vivo tumor growth and tumor microenvironment, we used a syngeneic and orthotopic mouse model of ovarian cancer in immunocompetent animals. We transduced a mouse tumor cell line (MOV1) with Katushka fluorescent protein (MOV1KAT) and here we show its orthotopic implantation in ovary and in vivo imaging.

浆液性卵巢癌原位模型在免疫小鼠，为在体内肿瘤显像和肿瘤免疫反应的监测

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

免疫与感染

Surface-enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detecting Microscopic Ovarian Cancer via Folate Receptor Targeting

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic spread has already occurred. However, ovarian cancer has a unique pattern of metastatic spread, in that tumor implants are initially contained within the peritoneal cavity. This feature could enable, in principle, the complete resection of tumor implants with curative intent. Many of these metastatic lesions are microscopic, making them hard to identify and treat. Neutralizing such micrometastases is believed to be a major goal towards eliminating tumor recurrence and achieving long-term survival. Raman imaging with surface enhanced resonance Raman scattering nanoprobes can be used to delineate microscopic tumors with high sensitivity, due to their bright and bioorthogonal spectral signatures. Here, we describe the synthesis of two &#39;flavors&#39; of such nanoprobes: an antibody-functionalized one that targets the folate receptor &#8212; overexpressed in many ovarian cancers &#8212; and a non-targeted control nanoprobe, with&#160;distinct spectra. The nanoprobes are co-administered intraperitoneally to mouse models of metastatic human ovarian adenocarcinoma. All animal studies were approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center. The peritoneal cavity of the animals is surgically exposed, washed, and scanned with a Raman microphotospectrometer. Subsequently, the Raman signatures of the two nanoprobes are decoupled using a Classical Least Squares fitting algorithm, and their respective scores divided to provide a ratiometric signal of folate-targeted over untargeted probes. In this way, microscopic metastases are visualized with high specificity. The main benefit of this approach is that the local application into the peritoneal cavity &#8212; which can be done conveniently during the surgical procedure &#8212; can tag tumors without subjecting the patient to systemic nanoparticle exposure. False positive signals stemming from non-specific binding of the nanoprobes onto visceral surfaces can be eliminated by following a ratiometric approach where targeted and non-targeted nanoprobes with distinct Raman signatures are applied as a mixture. The procedure is currently still limited by the lack of a commercial wide-field Raman imaging camera system, which once available will allow for the application of this technique in the operating theater.

Ovarian cancer forms metastases throughout the peritoneal cavity. Here, we present a protocol to make and use folate-receptor targeted surface-enhanced resonance Raman scattering nanoprobes that reveal these lesions with high specificity via ratiometric imaging. The nanoprobes are administered intraperitoneally to living mice, and the derived images correlate well with histology.

表面增强共振拉曼散射纳米探针比比法检测微卵巢癌的意义上

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic ...

An Orthotopic Mouse Model of Ovarian Cancer using Human Stroma to Promote Metastasis

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant transgenic mouse models of ovarian cancer exist, these mice are expensive and take a long time to develop tumors. Intraperitoneal injection xenograft models lack human stroma and do not accurately model ovarian cancer metastasis. Even patient derived xenografts (PDX) do not fully recapitulate the human stromal microenvironment as serial PDX passages demonstrate significant loss of human stroma. The ability to easily model human ovarian cancer within a physiologically relevant stromal microenvironment is an unmet need. Here, the protocol presents an orthotopic ovarian cancer mouse model using human ovarian cancer cells combined with patient-derived carcinoma-associated mesenchymal stem cells (CA-MSCs). CA-MSCs are stromal progenitor cells, which drive the formation of the stromal microenvironment and support ovarian cancer growth and metastasis. This model develops early and diffuses metastasis mimicking clinical presentation. In this model, luciferase expressing ovarian cancer cells are mixed in a 1:1 ratio with CA-MSCs and injected into the ovarian bursa of NSG mice. Tumor growth and metastasis are followed serially over time using bioluminescence imaging. The resulting tumors grow aggressively and form abdominal metastases by 14 days post injection. Mice experienced significant decreases in body weight as a marker of systemic illness and increased disease burden. By day 30 post injection, mice met endpoint criteria of &gt;10% body weight loss and necropsy confirmed intra-abdominal metastasis in 100% of mice and 60%-80% lung and parenchymal liver metastasis. Collectively, orthotopic engraftment of ovarian cancer cells and stroma cells generates tumors that closely mimic the early and diffuse metastatic behavior of human ovarian cancer. Furthermore, this model provides a tool to study the role of ovarian cancer cell: stroma cell interactions in metastatic progression.

Orthotopic engraftment of ovarian cancer cells mixed with human stromal cells provides a mouse model that exhibits rapid, diffuse metastatic behavior that is characteristic of human ovarian cancer. This model also allows for the study of tumor cell and stromal cell interactions, as well as their role in tumor progression and metastasis.

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant ...

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

Uterine cancers can be studied in mice due to the ease of handling and genetic manipulation in these models. However, these studies are often limited to assessing pathology post-mortem in animals euthanized at multiple time points in different cohorts, which increases the number of mice needed for a study. Imaging mice in longitudinal studies can track the progression of disease in individual animals, reducing the number of mice needed. Advances in ultrasound technology have allowed for the detection of micrometer-level changes in tissues. Ultrasound has been used to study follicle maturation in ovaries and xenograft growth but has not been applied to morphological changes in the mouse uterus. This protocol examines the juxtaposition of pathology with in vivo imaging comparisons in an induced endometrial cancer mouse model. The features observed by ultrasound were consistent with the degree of change seen by gross pathology and histology. Ultrasound was found to be highly predictive of the observed pathology, supporting the incorporation of ultrasonography into longitudinal studies of uterine diseases such as cancer in mice.

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

This protocol describes a method for monitoring the progression of morphological changes over time in the uterus in an inducible mouse model of endometrial cancer using ultrasound imaging with correlation to gross and histological changes.

小鼠中Pax8定向缺失肿瘤抑制因子 Arid1a 和 Pten 的子宫内膜癌进展的非侵入性超声评估

Uterine cancers can be studied in mice due to the ease of handling and genetic manipulation in these models. However, these studies are often limited to ...

建立卵巢癌-网膜相互作用的 3D 离体模型

An Ex Vivo Model of Ovarian Cancer Peritoneal Metastasis Using Human Omentum

Ovarian cancer is the deadliest gynecologic malignancy. The omentum plays a key role in providing a supportive microenvironment to metastatic ovarian cancer cells as well as immune modulatory signals that allow tumor tolerance. However, we have limited models that closely mimic the interaction between ovarian cancer cells and adipose-rich tissues. To further understand the cellular and molecular mechanisms by which the omentum provides a pro-tumoral microenvironment, we developed a unique 3D ex vivo model of cancer cell-omentum interaction. Using human omentum, we are able to grow ovarian cancer cells within this adipose-rich microenvironment and monitor the factors responsible for tumor growth and immune regulation. In addition to providing a platform for the study of this adipose-rich tumor microenvironment, the model provides an excellent platform for the development and evaluation of novel therapeutic approaches to target metastatic cancer cells in this niche. The proposed model is easy to generate, inexpensive, and applicable to translational investigations.

作者聚焦:用于研究癌症-脂肪微环境相互作用的高级离体模型 

This protocol describes the establishment of a three-dimensional (3D) ex vivo model of cancer cell-omentum interaction. The model provides a platform for elucidating pro-tumor mechanisms within the adipose niche and for testing novel therapies.

基于人网膜的卵巢癌腹膜转移离 体 模型

Ovarian cancer is the deadliest gynecologic malignancy. The omentum plays a key role in providing a supportive microenvironment to metastatic ovarian ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

生物学

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 ...

小鼠模型的非侵入性成像检测和监测卵巢癌复发

摘要

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此视频中的章节

原发肿瘤发展小鼠实验模型和腹膜转移

浆液性卵巢癌原位模型在免疫小鼠，为在体内肿瘤显像和肿瘤免疫反应的监测

表面增强共振拉曼散射纳米探针比比法检测微卵巢癌的意义上

An Orthotopic Mouse Model of Ovarian Cancer using Human Stroma to Promote Metastasis

小鼠中Pax8定向缺失肿瘤抑制因子 Arid1a 和 Pten 的子宫内膜癌进展的非侵入性超声评估

基于人网膜的卵巢癌腹膜转移离体模型

基于人网膜的卵巢癌腹膜转移离体模型

基于人网膜的卵巢癌腹膜转移离体模型

活体成像和治疗卵巢癌的原位小鼠模型

腹膜内卵巢癌转移的定量

小鼠模型的非侵入性成像检测和监测卵巢癌复发

摘要

探索更多视频

此视频中的章节

原发肿瘤发展小鼠实验模型和腹膜转移

浆液性卵巢癌原位模型在免疫小鼠，为在体内肿瘤显像和肿瘤免疫反应的监测

表面增强共振拉曼散射纳米探针比比法检测微卵巢癌的意义上

An Orthotopic Mouse Model of Ovarian Cancer using Human Stroma to Promote Metastasis

小鼠中Pax8定向缺失肿瘤抑制因子 Arid1a 和 Pten 的子宫内膜癌进展的非侵入性超声评估

基于人网膜的卵巢癌腹膜转移离 体 模型

基于人网膜的卵巢癌腹膜转移离 体 模型

基于人网膜的卵巢癌腹膜转移离 体 模型

活体成像和治疗卵巢癌的原位小鼠模型

腹膜内卵巢癌转移的定量

基于人网膜的卵巢癌腹膜转移离体模型

基于人网膜的卵巢癌腹膜转移离体模型

基于人网膜的卵巢癌腹膜转移离体模型