The Satchi-Fainaro research laboratory is partially supported by The Association for International Cancer Research (AICR), German-Israel Foundation (GIF), THE ISRAEL SCIENCE FOUNDATION (Grant No. 1309/10), Swiss Bridge Award, and by grants from the Israeli National Nanotechnology Initiative (INNI), Focal Technology Area (FTA) program: Nanomedicine for Personalized Theranostics, and by The Leona M. and Harry B. Helmsley Nanotechnology Research Fund.

注:所有动物的程序获得批准，并符合医学实验动物管理和使用委员会的特拉维夫大学萨克勒学校（IACUC）的标准执行。 1.空调媒体准备<ol><li>种子2×10 6的U-87 MG细胞在10cm的培养皿中的体积8毫升Dulbecco改进的Eagle培养基（DMEM），补充有10％胎牛血清（FBS）和1％青霉素/链霉素/制霉菌素溶液。 </li><li>孵育细胞在37 °℃; 5％CO 2，直到48小时实现全面融合。 </li><li>使用10毫升注射器，撤回所有介质从板和过滤通过0.45微米的过滤入15ml管中。 </li><li>未经洗涤细胞板，加入1 ml胰蛋白酶溶液（0.25％），并培育。 </li><li>当所有的细胞分离，使用至少5毫升的DMEM补充有10％FBS的灭活胰蛋白酶和预型体细胞计数。 </li><li<html> &gt;计数使用血球或其他细胞计数装置细胞的数量，并根据所使用的计数方法计算细胞的数目。 注:将在后面以正常化的CM的浓度被使用。 </li><li>所述CM转移到玻璃圆底烧瓶中并浓缩，在高真空旋转蒸发。为了加快该过程，增加浴温至37℃，如果需要的话。继续下去，直到CM达到糊状的质地。 </li><li>悬浮从每个培养皿中的400微升生理盐水CM。当使用一个以上的板，可使用等效的体积。例如，对于2培养板中，使用800微升。用每块板的4只小鼠。 </li><li>为了比较不同的细胞系或治疗，根据在步骤1.6接收到的细胞的比率调整音量。例如，如果一个板包含1.5倍以上的细胞，添加200μl的盐水，以预先存在的400微升。这将是调节的CM </li></ol>标题"&gt; 2。ECM-模仿硅胶塞接种到小鼠<ol><li>接种前的ECM-模仿凝胶到小鼠体内，准备以下材料: <ol><li>生长因子减少苯酚的无ECM-模拟物凝胶（解冻过夜，在实验前一天，在4℃下在冰上）。 </li><li>电动剃须刀。 </li><li>氯胺酮和甲苯噻嗪以麻醉小鼠。 </li><li>冰冷的1000微升无菌针头。 </li><li>冰冷1ml注射器和27G的针头。 </li></ol></li><li>准备每只小鼠含有80微升调节的CM 1.5ml的Eppendorf管中，并保持在冰上。 </li><li>向每个管中，加入600微升冰冷的生长因子被解冻过夜，在4℃，在冰上减少苯酚的无ECM-模拟凝胶。对ECM-模拟凝胶是液体在4℃，并固化在体温;为了防止对ECM-模仿凝胶从固化，用冰冷1000微升的提示。混合管仔细不会形成气泡，并保持在冰上。 </li><li>麻醉6周老B使用氯胺酮（100毫克/千克）和赛拉嗪（12毫克/千克）ALB / c小鼠。根据在开始实验前的机构动物护理和使用委员会， 即确认适当麻醉。动物已经达到麻醉自己正确的飞机时，他们不再以坚定的脚趾捏响应。 </li><li>剃鼠的腹部和消毒使用外科擦洗/醇。 </li><li>使用冷1ml注射器和27G的针来注入Eppendorf管皮下的内容。进行注射非常缓慢，以防止泡沫的形成。一旦所有卷管理，不弹出针几分钟，直至凝固发生。 </li></ol> 3.评估ECM-模仿硅胶塞血管化利用超声注:这部分协议要求操作超声成像系统，或经授权的技术人员的协助下以前的知识。 <ol><li>在开始之前，必须在Following材料和工具准备: <ol><li>电动剃须刀。 </li><li>脱毛霜。 </li><li>氯胺酮和甲苯噻嗪以麻醉小鼠。 </li><li>非针对性的微泡。 </li><li>激活设备。 </li><li>美国成像系统，其包括一个55兆赫708探针和3D获取显微。 </li></ol></li><li>插头后三周接种内测量血管: 注:在U-87 MG型动物模型，3周被发现代表了成像的理想时间点。根据不同的设置，校准从接种后塞几天开始血管动力学。 <ol><li>麻醉用氯胺酮（100毫克/千克）和赛拉嗪（12毫克/公斤）小鼠。根据在开始实验前的机构动物护理和使用委员会， 即确认适当麻醉。动物已经达到麻醉自己正确的飞机时，他们不再以坚定的脚趾捏响应。 </li><li>剃小鼠的腹部区域，并用脱毛霜。 </li><li>固定的小鼠仰卧在微操纵器的阶段。 </li><li>使用含生理盐水1ml注射器和一个地下30针，将针尾静脉内，固定针头牢固，松动的注射器。 </li><li>建立起来用于对比度3D模式的图像采集会话中: <ol><li>按"3D"初始化3D马达阶段，并将换能器在所需的扫描区域的中间。 </li><li>按"3D"，并输入扫描距离和步长，然后按"扫描"。 </li><li>确保整个插头被扫描中显示的3D图像数据。如果不是这样，调整振子，以使整个插头是在扫描和重复步骤3.5.2可见。 </li><li>按"对比度"，推动了时间增益补偿（TGC）控制到左边使图像变暗。 </li></ol></li><li>激活使用小瓶混合机是DESI微泡gnated对微泡的活化为45秒。 </li><li>混合在1ml注射器将50μl的微泡用50μl生理盐水。 </li><li>更换含生理盐水松动注射器含有活性微泡的注射器。注入稀释微泡入尾静脉，小鼠的，等待几秒钟的气泡，以达到稳定状态，然后获得插头的3D造影增强电影回放，使用3D采集电机: <ol><li>按"3D"，然后选择"扫描"，以获得3D对比度增强图像，然后按"电影商店"保存图像数据。 </li><li>按"3D"，然后单击"消灭3D"，以销毁的微泡。 </li><li>按"3D"再选择"扫描"收购后破坏电影圈，然后按"电影商店"。 </li></ol></li><li>生成的叠加图像帧之前和之后的破坏微泡: <ol><li>在＆＃8220;参考设置"在屏幕的左侧点击"创建引用"。 </li><li>按"学习管理"，打开获得的预破坏电影圈。 </li><li>单击"进程电影"生成绿色的对比叠加，然后单击"加载到3D"。 </li></ol></li><li>使用美国成像软件，以定量的插头内的血液量得到的三维图像: <ol><li>按"扫描/冻结"，然后按"模式设置"。 </li><li>单击"音量"，然后选择"并行"。 </li><li>通过分割一系列轮廓创建目标组织的三维体积。 </li><li>点击"F​​inish"完成分割。 注:在造影剂的量将在屏幕的左下侧显示。 </li></ol></li><li>在程序结束时，小鼠放置在温暖，干净，干燥，安静的环境，远离其他动物。使用市售的手术加热垫或白炽灯（50-75瓦），12-14英寸的距离放置啮齿动物提供温暖。连续监测小鼠，直到保持直立的姿势和行走正常。 </li></ol></li></ol> 4.评价ECM-模仿硅胶塞脉管形态使用纤维状的共聚焦显微内镜注:这部分协议的执行紧随美国成像，它需要操作纤维质的共聚焦显微内镜成像系统，或经授权的技术人员的协助下以前的知识。 <ol><li>开始前，有下列材料和工具准备: <ol><li>手术工具，如镊子，剪刀和可吸收缝合线。 </li><li>的FITC缀合的葡聚糖（70 kDa）的。 </li><li>校准溶液（3试管各含有过氧化氢，水或荧光溶液）。 </li><li>所述成像系统和一个探头适于血管根据制造商的成像。 </li></ol></li><li>麻醉用氯胺酮（100毫克/千克）和赛拉嗪（12毫克/公斤）小鼠。 </li><li>辖四，经尾静脉，200微升10毫克/毫升的FITC-标记的葡聚糖。 </li><li>使用外科擦洗/酒精消毒腹部区域，并从插头施加一个小的切口大约1厘米。 </li><li>将显微内镜探针上的插头的顶部轻轻同时保持插头完好无损。 </li><li>收购: <ol><li>一旦探针被设置在插头上时，按使用脚踏板"开始激光"。荧光应该显示在屏幕上。 </li><li>按"记录"在屏幕上或&#39;选择&#39;使用的脚踏板获得短片（最长30秒）。 </li><li>电影之间，通过将尖端的含管水冲洗探头。 </li><li>完成后，取下探头，并用干净的棉签尖端。 </li></ol></li><li>分析平均值已经SSEL直径: <ol><li>点击"检测微血管"图标在上面板以打开容器检测模块窗口。 </li><li>点击"首选项"，并标注在"统计的利益"和"显示直方图条"中的"显示模式"，"平均血管直径"。 </li><li>点击"检测"，以显示选定的测量。 </li></ol></li><li>为了分析在邻近血管的区域的荧光信号强度: <ol><li>请点击左侧面板中的"创建圈子的投资回报率"，创造在感兴趣的领域了一圈。 </li><li>点击"范围内的投资回报率计算直方图"计算与本区域的值。 </li></ol></li></ol>小鼠5.后处理程序<ol><li>在每个成像过程的结束，将小鼠在一个温暖，干净，干燥，安静的环境，远离其他动物。使用市售-一个提供温暖vailable手术加热垫或白炽灯（50-75 W），放置在12-14远离啮齿动物。连续监测小鼠，直到保持直立的姿势和行走正常。 </li><li>对于塞"血管额外的时间点进一步分析，应用以下条件恢复: <ol><li>使用缝合吸收缝合线的小切口。 </li><li>给予小鼠镇痛如丁丙诺啡（1毫克/千克）经皮下，并返回到单独的笼子中。 </li><li>遵守所有的动物，以确定是否进一步止痛治疗是必要的和重新评估的日常监测和评估遇险。 </li></ol></li><li>在终点的实验中，小鼠安乐死通过首先施加异氟烷，随后通过颈脱位。 </li><li>如果额外的影像将在初始成像步骤周进行，所有的程序应保持无菌越好。应进行外科手术的皮肤擦洗，消毒娃之三和无菌棉尖端应当用来清洁显微内镜探针，并且探针应该使用无菌水各动物之间进行消毒。 </li></ol>

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The authors have nothing to disclose.

结合互补的成像方法允许在不同的血管生成组件的微血管密度，功能和形态的信息的获取。在ECM-模拟凝胶塞装载的CM产生一个肿瘤微环境，这是从其他细胞群中分离，如内皮细胞，该被招募并渗出到插头。 通过执行，并行地，微泡造影美国成像和纤维状共聚焦显微内镜成像，我们可以得出结论，一池由快速生长的血管生成的肿瘤产生的细胞分泌的因素，而不癌细胞本身，不仅能够招募血管，但还形成与EPR典型特征功能血管。此外，这种方法允许分泌的因子在肿瘤微环境的影响，以及细胞 - 细胞相互作用之间的距离。 信息获得来自美国和纤维状共聚焦然而显微内镜经受成像技术的限制。例如，在使用纤维质共聚焦显微内镜成像，这是不可能的图像的单个特定血管随时间，或甚至在同一地区的插头。 对ECM-模拟凝胶塞测定是比较容易进行，这是与ECM-模拟凝胶装载的CM时持续。然而，应在考虑采取的一个关键问题是要CM ECM-模拟凝胶的比例。高容量CM可能阻止ECM-模仿凝胶固化。因此，CM量不应超过对ECM-模拟凝胶的约13％。 这种方法可以用于不同的实验系统和应用程序进行调整。例如，它可以被用来评估抗血管生成药物的装载的CM从前后处理的细胞。此外，其他的细胞系可根据这些实验设置使用。然而，要求是为了校准到一个pply最佳CM浓度。而且，对于成像的理想时间点，应根据各模型系统进行了优化。

基质胶塞血管生成测定首先由Kibbey 等人于1992年，在那里它被用来由肽SIKVAV（丝氨酸-异亮氨酸-赖氨酸-缬氨酸-丙氨酸-缬氨酸）1来评估血管生成的刺激说明。相对于其他的体内血管发生测定法，像鼠标角膜血管发生或鸡胚尿囊膜测定法，该测定法是比较容易进行2。注入的ECM-模拟凝胶可包含细胞，促血管生成或抗血管生成的化合物和/或因素。当评估抗血管生成化合物的活性，所述插头通常包含的血管内皮生长因子（VEGF）和成纤维细胞生长因子（FGF）的混合物中，并且所述抗血管生成物质可直接施用到插头或全身三4。 对ECM-模拟凝胶皮下注射那里分钟内固化。血管招聘中产生的插头评估。典型ý通过以下注射荧光素异硫氰酸（FITC）测定血红蛋白水平内塞，通过荧光信号的测量进行标记的葡聚糖，通过组织学切片内皮特异性标记物的免疫染色或通过荧光激活细胞分选（FACS）2,5， 6。但是，此评价只允许终点分析，缺乏有关的血管的功能和形态信息。此外，血浆量的测量结果，无论是由血红蛋白水平或通过使用葡聚糖-FITC是一个指标，可能会产生误导，因为血液含量是受血管的大小和血停滞池的程度。这是特别重要的，当招募血管的特征在于增强的渗透性和保留（EPR）效果7。 我们在此通过结合两个互补成像方式提出了一种新的，更精确的，方法的招募血管可视化。 HIGH-分辨率微泡超声造影（美国）联合活fibred共焦显微内镜不仅能够提供有关血管密度，同时也对自己的形态和功能的信息。此外，这种分析可以在几个时间点，从而使监控血管发生动力学来执行。美国是一种广泛使用的成像模态而具有以下静脉内（IV）注射仍然存在只在血管隔室8-11微泡的高空间分辨率的血管成像。微泡是产生一个强回声信号时与美国脉冲激发，因此可作为一个良好的造影剂气体填充的微泡。使用下列微泡破坏美国成像系统软件中获取的插头的3D图像。所产生的图像是由前，后破坏微泡的图像帧组成的，并反映在彩色视频强度差。这些叠加图像由软件自动显示。因此，插头内的功能血管的百分比可以量化。纤维状共聚焦显微内镜高分辨率作为通过血管形态报告的补充成像方式。在这种微创方法，图像采集的FITC标记的右旋糖酐12静脉给药后。荧光聚合物染料的血液，从而作为"造影剂"的血管形态和血流。此外，由于所注入的聚合物是在70 kDa的大小，它只能交叉渗漏血管如肿瘤血管中找到。这将导致从血管外的FITC-标记的葡聚糖的高背景。因此，纤维状的共聚焦显微内镜可用于可视化的EPR效应典型特征在肿瘤neovasculature-放大，泄漏的，高度缠结的船只，具有平端。 该系统模型descri床的U-87 MG人脑胶质瘤和休眠克隆13的血管生成潜力的比较。插头内血管进行评价后三周的ECM-模拟凝胶接种。结果表明，含有的CM从插头内的血管的U-87 MG快速增长的数量，密度和功能性，方面当与含有的CM从休眠肿瘤产生的细胞内塞血管相比肿瘤产生细胞显著增加。因此，研究者们能够得出这样的结论CM从隔离U-87 MG快速增长的肿瘤细胞产生含有较高水平的促血管生成因子，从休眠的肿瘤细胞产生的CM相比。这些因素刺激功能血管的形成通过积极地影响所有步骤的血管发生级联（ 即 ，增殖，出芽，迁移，最后，形成内皮细胞的管状结构）。

<table><tbody><tr><td>Rotary Vacuum Evaporator</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Growth-factors reduced phenol-free Matrigel</td><td>BD Bioscience</td><td>FAL356231</td><td>Thaw overnight, at 4 &amp;deg;C on ice</td></tr><tr><td>Depilatory cream</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Vevo2100 US imaging system</td><td>VisualSonics Inc.</td><td>-</td><td>Requires previous knowledge in operating or the assistance of an authorized technician</td></tr><tr><td>55 MHz 708 probe</td><td>VisualSonics Inc.</td><td>-</td><td>-</td></tr><tr><td>Vevo2100 imaging software</td><td>VisualSonics Inc.</td><td>-</td><td>-</td></tr><tr><td>VialMix</td><td>DEFINITY Imaging</td><td>VMIX</td><td>-</td></tr><tr><td>DEFINITY microbubbles</td><td>DEFINITY Imaging</td><td>DE4</td><td>Activate for 45 sec using VialMix before use</td></tr><tr><td>CellVizio (Fibered confocal endomicroscope)</td><td>Mauna Kea Technologies</td><td>-</td><td>Requires previous knowledge in operating or the assistance of an authorized technician</td></tr><tr><td>ProFlex MiniO/30 probe</td><td>Mauna Kea Technologies</td><td>-</td><td>-</td></tr><tr><td>70 kD FITC-labeled dextran</td><td>Sigma-Aldrich</td><td>46945</td><td>-</td></tr><tr><td>ImageCell software</td><td>Mauna Kea Technologies</td><td>-</td><td>-</td></tr><tr><td>Calibration Kit</td><td>Mauna Kea Technologies</td><td>-</td><td>-</td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium (DMEM)</td><td>Gibco Life Tecchnologies</td><td>41965-039</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Biological Industries</td><td>04-007-1A</td><td></td></tr><tr><td>Penicillin/streptomycin/nystatin solution</td><td>Biological Industries</td><td>03-032-1B</td><td></td></tr></tbody></table>

monitoring functionality and morphology of vasculature recruited by factors secreted by fast-growing tumor-generating cells

在小鼠中的皮下基质胶栓测定是用于亲和抗血管生成因子在体内的评价所选择的方法。在该方法中，期望的因素被引入到冷液体的ECM-模拟凝胶其中，皮下注射后，固化形成模仿癌环境的环境。这个矩阵允许的宿主细胞，如内皮细胞，和血管因此，形成的渗透。 这里我们提出了一种新的改性的基质胶塞测定法，其可被利用来说明由癌细胞分泌的因子池的血管生成潜力，而不是一个特定的因子（ 例如，bFGF及VEGF）或代理。含ECM-模拟凝胶插头用来引进主机（ 即鼠标）与分泌，以快速增长的肿瘤生成胶质母细胞瘤细 ​​胞的CM因素池。我们前面所述的血管生成潜在的广泛的比较的U-87 MG人类成胶质细胞瘤及其蛰伏衍生克隆，在该系统模型，示出在U-87 MG亲代细胞诱导的血管生成。所述CM通过过滤从任一细胞系的汇合组织培养板下面48小时培养收集介质制备。因此，它仅包含由细胞分泌的因素，而不细胞本身。这里描述的是两个成像模态的组合，微泡超声造影成像和活体纤维质共焦显微内镜，为准确，实时的程度，形态表征插头内新形成的血管和功能。

植入ECM-模拟胶栓从快速增长的血管生成肿瘤产生的细胞系U-87 MG，导致广泛的血管生成CM。这种脉管可以通过使用两个互补的成像方法进行了广泛探索。 微泡造影剂增强成像美国由表示内的U-87 MG的CM加载插头，而不是检测不到血流含有从休眠肿瘤产生细胞的CM插头内（ 图1）广泛的血流量显示的功能性血管进入插头广泛招募。 这些新形成的血管的形态是由纤维状共聚焦显微内镜（ 图2）强调。插头装有U-87 MG CM内血管形态呈现典型的增强通透性和保留（EPR）效果大分子如葡聚糖-FITC在70 kDa的尺寸用在这里。血管被扩大和高度TA<html> ngled，非连续和流动不畅（ 图2A，C）。另外，血液渗漏，观察到周围的环境中，如由血管（ 图2B）以外的高荧光信号。 <img alt="图1" fo:content-width="6.5in" src="/files/ftp_upload/51525/51525fig1highres.jpg" width="600" /> 图1:微泡造影成像美国继微静脉给药和销毁，含插头展览U-87 MG CM增加血管（绿色）。含CM从U-87 MG衍生休眠的 ​​肿瘤细胞产生插头作为对照。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51525/51525fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> "SRC ="/文件/ ftp_upload / 51525 / 51525fig2highres.jpg"WIDTH ="500"/&gt; 图2:光纤共聚焦显微成像。 （A）的70kDa的葡聚糖-FITC静脉给药，使血流量和血管形态的可视化。 U-87 MG CM含塞血管包括漏，扩大船只钝器结束。血管渗漏是由血管外荧光信号显示出来。在小鼠腹部正常脉管系统被用作对照。（B）中在相邻的血管的区域的荧光信号强度。荧光信号由一个谱例举从红色到白色。暗红色代表低信号强度，而白色代表高荧光信号。血管的U-87 MG的CM含有插头内的U-87 MG的CM含有插头显示血管外的清晰的荧光信号，而没有任何信号被示出正常血管外。（C）的平均血管直径（MVD）（22.4微米）是标志意义ntly较高相比，正常血管（6.7微米）的MVD。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51525/51525fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Monitoring Functionality and Morphology of Vasculature Recruited by Factors Secreted by Fast-growing Tumor-generating Cells at JoVE.com

Monitoring Functionality and Morphology of Vasculature Recruited by Factors Secreted by Fast-growing Tumor-generating Cells

We describe a matrigel plug assay to illustrate angiogenic potential of a pool of factors secreted by cancer cells using two complementary imaging modalities, ultrasound and endomicroscopy. The matrigel, an extracellular matrix (ECM)-mimic gel, is utilized to introduce the host (mouse) with angiogenic factors secreted to the conditioned media (C.M.).&#160;

监控功能和血管形态由快速增长的肿瘤产生细胞分泌的因素招募

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired ...

monitoring-functionality-morphology-vasculature-recruited-factors

Nanyang Technological University

Research

JoVE Journal

Biology

9.7K 次观看.  Tel Aviv University. We describe a matrigel plug assay to illustrate angiogenic potential of a pool of factors secreted by cancer cells using two complementary imaging modalities, ultrasound and endomicroscopy. The matrigel, an extracellular matrix (ECM)-mimic gel, is utilized to introduce the host (mouse) with angiogenic factors secreted to the conditioned media (C.M.). 

视频: Monitoring Functionality and Morphology of Vasculature Recruited by Factors Secreted by Fast-growing Tumor-generating Cells

Assessing Tumor Microenvironment of Metastasis Doorway-Mediated Vascular Permeability Associated with Cancer Cell Dissemination using Intravital Imaging and Fixed Tissue Analysis

The most common cause of cancer related mortality is metastasis, a process that requires dissemination of cancer cells from the primary tumor to secondary sites. Recently, we established that cancer cell dissemination in primary breast cancer and at metastatic sites in the lung occurs only at doorways called Tumor MicroEnvironment of Metastasis (TMEM). TMEM doorway number is prognostic for distant recurrence of metastatic disease in breast cancer patients. TMEM doorways are composed of a cancer cell which over-expresses the actin regulatory protein Mena in direct contact with a perivascular, proangiogenic macrophage which expresses high levels of TIE2 and VEGF, where both of these cells are tightly bound to a blood vessel endothelial cell. Cancer cells can intravasate through TMEM doorways due to transient vascular permeability orchestrated by the joint activity of the TMEM-associated macrophage and the TMEM-associated Mena-expressing cancer cell. In this manuscript, we describe two methods for assessment of TMEM-mediated transient vascular permeability: intravital imaging and fixed tissue immunofluorescence. Although both methods have their advantages and disadvantages, combining the two may provide the most complete analyses of TMEM-mediated vascular permeability as well as microenvironmental prerequisites for TMEM function. Since the metastatic process in breast cancer, and possibly other types of cancer, involves cancer cell dissemination via TMEM doorways, it is essential to employ well established methods for the analysis of the TMEM doorway activity. The two methods described here provide a comprehensive approach to the analysis of TMEM doorway activity, either in na&#239;ve or pharmacologically treated animals, which is of paramount importance for pre-clinical trials of agents that prevent cancer cell dissemination via TMEM.

We describe two methods for assessing transient vascular permeability associated with tumor microenvironment of metastasis (TMEM) doorway function and cancer cell intravasation using intravenous injection of high-molecular weight (155 kDa) dextran in mice. The methods include intravital imaging in live animals and fixed tissue analysis using immunofluorescence.

利用生命内成像和固定组织分析评估与癌细胞传播相关的转移门道介导血管渗透性的肿瘤微环境

The most common cause of cancer related mortality is metastasis, a process that requires dissemination of cancer cells from the primary tumor to secondary ...

科研

癌症研究

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

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

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

实时成像在乳腺癌小鼠模型中的肿瘤微环境对药物的反应

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

医学

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

一种新型的高分辨率<em&gt;在体内</em&gt;成像技术研究肿瘤生长及治疗颅内结构的动态响应

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

制备基于微流体的血管化人类微肿瘤模型

Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical cancer research and therapeutic development.&#160;To overcome this problem, we have developed the vascularized microtumor (VMT), or tumor chip, a microphysiological system that realistically models the complex human tumor microenvironment. The VMT forms de novo within a microfluidic platform by co-culture of multiple human cell types under dynamic, physiological flow conditions. This tissue-engineered micro-tumor construct incorporates a living perfused vascular network that supports the growing tumor mass just as newly formed vessels do in vivo. Importantly, drugs and immune cells must cross the endothelial layer to reach the tumor, modeling in vivo physiological barriers to therapeutic delivery and efficacy. Since the VMT platform is optically transparent, high-resolution imaging of dynamic processes such as immune cell extravasation and metastasis can be achieved with direct visualization of fluorescently labeled cells within the tissue. Further, the VMT retains in vivo tumor heterogeneity, gene expression signatures, and drug responses. Virtually any tumor type can be adapted to the platform, and primary cells from fresh surgical tissues grow and respond to drug treatment in the VMT, paving the way toward truly personalized medicine. Here, the methods for establishing the VMT and utilizing it for oncology research are outlined. This innovative approach opens new possibilities for studying tumors and drug responses, providing researchers with a powerful tool to advance cancer research.

作者聚焦：创建人类血管化微肿瘤作为转化癌症研究的模型 

This protocol presents a physiologically relevant tumor-on-a-chip model to perform high-throughput basic and translational human cancer research, advancing drug screening, disease modeling, and personalized medicine approaches with a description of loading, maintenance, and evaluation procedures.

建立用于癌症研究的生理性人血管化微肿瘤模型

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

的肿瘤特异性内皮细胞的分离与培养扩展

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

实时多细胞动力学延长时间推移活体成像在肿瘤微环境

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

基于一个基质胶管形成实验，以评估对肿瘤细胞的血管生成活性

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

生物学

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

监控功能和血管形态由快速增长的肿瘤产生细胞分泌的因素招募

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利用生命内成像和固定组织分析评估与癌细胞传播相关的转移门道介导血管渗透性的肿瘤微环境

实时成像在乳腺癌小鼠模型中的肿瘤微环境对药物的反应

一种新型的高分辨率

建立用于癌症研究的生理性人血管化微肿瘤模型

建立用于癌症研究的生理性人血管化微肿瘤模型

建立用于癌症研究的生理性人血管化微肿瘤模型

的肿瘤特异性内皮细胞的分离与培养扩展

实时多细胞动力学延长时间推移活体成像在肿瘤微环境

基于一个基质胶管形成实验，以评估对肿瘤细胞的血管生成活性

应用先进的背皮窗室对转基因荧光小鼠肿瘤相关血管的活体显微镜观察