Name: extended time-lapse intravital imaging of real-time multicellular dynamics in the tumor microenvironment
Uploaded: 2016-06-12T14:00:00.000+00:00
Duration: 8 min 24 s
Description: Watch this Scientific Journal Video about Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment at JoVE.com

这项研究是由美国国防部的乳腺癌研究计划下授予号（ASH，W81XWH-13-1-0010），NIH CA100324，PPG CA100324，和综合成像计划的支持。

中描述的所有程序必须按照与使用脊椎动物，包括医药机构动物护理和使用委员会的爱因斯坦医学院的事先批准的准则和规定进行。 1.生成荧光标记的肿瘤及肿瘤相关巨噬细胞<ol><li>通过杂交自发，原地，遗传工程小鼠乳腺癌模型中，小鼠乳腺肿瘤病毒长末端重复与荧光报告[ 即转基因小鼠驱动多瘤中间T抗原（MMTV-PyMT），增强型绿色荧光产生的荧光标记的肿瘤细胞蛋白（EGFP），增强型青色荧光蛋白（ECFP）或者Dendra2] -8,9-。 </li><li>在PyMT动物荧光标记的巨噬细胞用荧光通过穿越myeloid-和巨噬细胞特异性荧光记者遗传工程小鼠模型[即 MacGr标记的肿瘤细胞EEN 15或MacBlue小鼠CSF1R -GAL4VP16 / UAS-ECFP 16]。 </li><li>可替代地，通过荧光标记PyMT肿瘤细胞（同基因），人乳腺癌细胞系或原代人类患者来源的肿瘤（异种移植物）11,17的原位移植产生荧光标记的肿瘤。 <ol><li>植入荧光标记PyMT肿瘤细胞与荧光蛋白标记的骨髓细胞同系小鼠以生成具有荧光标记的肿瘤细胞和骨髓细胞13的动物。 </li></ol></li><li>注入100微升的10毫克/公斤的荧光70 kDa的葡聚糖通过静脉内（IV）尾静脉进入重症联合免疫缺陷（SCID）小鼠与人细胞系或来源于患者的原发肿瘤荧光标记的巨噬细胞2小时之前开始的异种移植肿瘤的活体成像。 </li></ol> 2.显微镜设置和成像准备注:此过程描述的设立对多光子显微镜8活体成像。 <ol><li>打开所有显微镜和激光组件，包括双光子激光器和检测器。 </li><li>打开加热框到30℃预温阶段。这个步骤是用于维持动物的生理温度是至关重要的。 </li><li>有关倒置显微镜的地方在显微镜舞台上的定制阶段插入使用。自定义插入是"加工成适合在舞台插入空间和一个1厚铝"直径的通孔的中心成像的1/8片。 <ol><li>擦拭用70％乙醇和空气干燥的显微镜载物台和阶段插入。 </li><li>放置在20X，1.05 NA显微镜物镜一大滴的水，以保持与玻璃盖光学接触。 </li><li>地方盖玻璃（＃1.5厚度）在显微镜舞台上插入成像端口。安全与实验室胶带的地方。 </li><li>用打孔器打孔2厘米×2厘米的洞以灵活的橡胶垫，并将与在自定义阶段插入孔对准垫孔显微镜阶段的橡胶垫。 </li></ol></li></ol> 3.尾静脉导管的制备及试剂成像注射期间<ol><li>切聚乙烯（PE）管30厘米长。 </li><li>使用镊子，移动31克针金属针来回，直到它打破关闭塑料接头的。 </li><li>使用镊子，插入分离针的钝端进入PE管材。 </li><li>插入一个31克针进入PE管的另一端。 </li><li>填用无菌磷酸盐缓冲盐水（PBS）1毫升注射器，并将其插入到31克针和刷新所有空气从管和针用PBS中。 PBS用于维护水合和血液的渗透压9,18，也可以使用然而等渗盐水。 </li><li>填用200μl的3毫克1毫升注射器/ kg的155 kDa的葡聚糖 - 四甲基罗丹明（TMR）或血管标记的量子点。 </li><li>制备任何其它可注射的蛋白质，例如在1毫升注射器置于冰上血管内皮生长因子A（VEGFA 165）的一个165氨基酸同种型。注入0.2毫克/千克VEGFA 165 19以0.05毫克/毫升的浓度。 </li></ol> 4.插入尾留置静脉置管<ol><li>使用感应腔室与O 2作为载气的5％异氟烷放置麻醉下的动物。 </li><li>鼠标转移到外科平台一旦完全麻醉，并且不向脚趾捏响应。 </li><li>开异氟烷麻醉线到外科平台，关闭麻醉线到感应腔室，并放置在动物的鼻子的鼻锥体。从5％减小异氟醚为2.5％。 </li><li>应用眼药膏鼠标，以防止在麻醉过程中干涩的眼睛。 </li><li>热的加热灯下动物另外的2分钟，以增加流通尾巴作为循环深麻醉放缓。 </li><li>消毒用70％乙醇的小鼠尾静脉。 </li><li>插入从第3节中构建到动物的侧面尾静脉导管的31克针尖和推针在2-3毫米。 </li><li>向后拉动注射器见血，确保导管放置在尾静脉中。 </li><li>使用放置在针实验室带的1cm长，以代替平行针保持沿尾部的长度的静脉。 </li></ol> 5.皮瓣手术过程揭露乳腺肿瘤<ol><li>与手术平台上的动物拭肿瘤并用70％乙醇腹面。注意:如所描述的，不进行该过程完全在无菌条件下。对于长成像会议，其中感染可成为一个混杂因素，建议使用正确的无菌技术。 </li><li>用免缝勒镊子，提起腹侧皮肤，并用无菌剪刀使一个皮下切口沿腹中线大约为1厘米的长度。避免切口时刺破腹膜。 </li><li>轻轻切结缔组织附着的乳腺肿瘤从腹膜掉，露出乳房肿瘤。 </li><li>用剪刀和镊子，轻轻取出，并从肿瘤的暴露表面切去筋膜和脂肪，同时保持肿瘤脉管系统的完整性和减少出血。这一步骤是在维持肿瘤的肿瘤架构和脉管供应的关键。 </li></ol> 6.动物准备显微镜<ol><li>动物转移到预热的成像级与暴露的肿瘤放置在盖玻片上的阶段插入在用于成像的显微镜物镜。照顾到与动物轻轻转移尾静脉导管，以便不去除针头。 </li><li>第广场Ë麻醉鼻锥在动物的鼻子，以保持麻醉盘以3％异氟醚。 </li><li>使肿​​瘤掌握在橡胶垫的孔，并与该玻璃盖玻片接触动物的位置。 </li><li>用两个橡胶垫到位轻轻握住肿瘤，并将其固定在显微镜阶段，实验室的磁带，以减少图像采集过程中的运动。 </li><li>填补用PBS橡胶垫的腔室，以保持组织水合和保持与盖玻片光学接触。 </li><li>开始脉搏血氧仪探头监测动物的生命体征。附加的夹子传感器动物的大腿。备选地，可以使用被配置为套在脖子上的套环。 </li><li>放置加热箱在动物保持30℃20。 </li><li>异氟醚的水平慢慢降低到0.5-1％维持麻醉，维持血流。 </li></ol> 7.图像采集和Fluoresc注射耳鼻喉科染料和注射蛋白质<ol><li>使用显微镜目镜聚焦于肿瘤的表面上的荧光肿瘤细胞。 </li><li>通过用流动的血管发现区域选择感兴趣的区域。感兴趣的区域的与流血管的选择是用于评估肿瘤血管的关键。 </li><li>一旦感兴趣区域已被选择切换显微镜到多光子成像模式。 </li><li>设置Z系列的上限和下限。通过使用焦点调整到目标移动到所需的开始位置和点击的Z位置"顶部"按钮组的z系列的上限。通过在二次谐波产生（SHG）信道的肿瘤表面可视化胶原纤维网络并当无细胞，但只胶原纤维是可见的观测确定的z系列的上限。 <ol><li>由物镜移动到所希望的成像深度设置Z系列的下限（typicaLLY 50 - 150微米），并单击Z位置"底部"按钮。 </li><li>通过键入所需的值到步长字段设置为步长。确定基于考虑分辨率和采集时间步长（通常为2微米，用于高分辨率三维重建和5微米其他）。 </li></ol></li><li>通过切换到时间推移面板和输入所需的经过时间进入时间推移字段中设置的时间推移的时间间隔。为获得最佳的时间分辨率的时间间隔设置为0秒，连续成像。注意:对于长期延时成像建议的时间间隔设置为10秒采集之间以补充目标浸没液体。 </li><li>一旦成像参数已经确定，缓缓注入155 kDa的葡聚糖TMR。 <ol><li>在尾静脉导管除去，用PBS注射器，用含155 kDa的葡聚糖的TMR注意不要引入任何气泡进入里注射器替换东北。 </li><li>缓缓注入155 kDa的葡聚糖的TMR到小鼠，用200μl的最大体积，并用含有PBS注射器更换注射器。关键的一步:慢慢进行注射，并采取预防措施，以避免让静脉以外的解决方案。 </li></ol></li><li>通过点击的Z-Stack和时间流逝按钮来抑制它们，然后点击录制按钮开始采集的Z堆叠延时成像。 </li><li>如果其它荧光葡聚糖， 即 10 kDa的葡聚糖-荧光素异硫氰酸酯（FITC），或蛋白质， 即 ，VEGFA 165，已事先准备，图像采集在= 0分钟开始开始后注入它们。 <ol><li>在尾静脉导管除去，用PBS注射器，用含10 kDa的葡聚糖-FITC或VEGFA 165注意不要引入任何气泡进入线注射器替换。 </li><li>通过尾静脉导管缓缓注入注射到小鼠和替换用含有PBS中的注射器的注射器。 </li></ol></li><li>每30-45分钟，慢慢注入50μl的PBS或生理盐水，以保持动物的水合。 </li></ol> 8.安乐死<ol><li>在图像采集的终止，安乐死的动物。 <ol><li>增加异氟烷％至5％。 </li><li>保持5％异氟醚下的动物，直到30秒它停止呼吸，从舞台上拆下后的动物。 </li><li>执行颈椎脱位，以确保完全安乐死。 </li></ol></li></ol> 9.图像处理<ol><li>获得在16位TIFF文件的图像在每个时间点每个单独的信道，并依次保存。 </li><li>执行频谱重叠（ 即绿色荧光蛋白和CFP），消除XY漂移和电影的使用ImageJ的中9条设立的方法时间推移序列产生的分离。进行高分辨率图像13的三维表面重建。 </li></ol>

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作者什么都没有透露。

在肿瘤微环境自发发生的细胞相互作用可以导致肿瘤细胞运动和血管内的变化。活肿瘤组织的高分辨率活体成像允许多细胞动力学，可以是高瞬态10,13,24的可视化。终点与分立时间点获取的可提供的在肿瘤微环境的过程的分子机制的基本信息体内测定或时间推移的图像。活体成像的研究已经被用于研究在肿瘤微环境中发生的数天的过程，生成对血管生成，肿瘤细胞迁移和响应新的见解对药物治疗25-30。但是，这些测定法可能无法捕获在肿瘤微环境瞬态事件的动力学因过程的时间尺度正在研究7,31。从不断的过程区分多个瞬态事件是一个显著的广告活体显微镜的优势。 在这个协议中描述的活体成像技术使得能够通过多个细胞谱系和结构的荧光标记在肿瘤微环境的细胞动力学的直接可视化。多光子显微镜允许使用超过300微米深的单光子共聚焦显微镜成像更深入常规进行24。的50的Z-栈采集- 100微米与2微米的步骤是足够高的分辨率，以产生细胞相互作用的三维重建，包括凸部使细胞使它们朝着在肿瘤微环境互相13和血管结构延伸。从隔时图像的序列的电影和三维重建允许细胞运动（速度和方向）的定量，并使用在注射染料，血管通透性的荧光信号强度的变化。 为了捕捉藻ontaneous细胞事件在单个序列捕获的个别图像的数目必须被优化以允许为图像采集的速度捕获的事件的动力学，但也涵盖了足够的物理空间来增加期间捕获任何个别事件的概率单延时序列。需要被考虑的空间成像参数包括:切片在Z堆叠的数量，视与取得的Z-堆栈和在Z堆叠个别切片之间的轴向距离场的数目。因此，在开始图像采集之前设置这些参数是协议的一个关键步骤。使用多光子显微镜，采集在Z系列30帧（500微米×500微米）的大约是60秒。由于观察到的肿瘤细胞血管内的持续时间可以是分13的数量级上，捕捉数的Z堆叠的三维重建血管内的，一个Z堆叠的采集时间是有限的吨Ø60秒。因此，被收购的视图与在Z堆叠多达30片单个字段。此外，由于细胞的三维重建被期望，Z-片之间的轴向步骤设定为2微米。这样就可以对细胞体积足够的信息，以便三维重建。这些参数必须根据所研究的现场活动的动力学使用的任何显微镜和图像处理软件进行优化。 显微镜舞台上和尾静脉静脉导管的橡胶垫的变形都既允许在成像时间显著增加。橡胶垫形成孔，以允许肿瘤的维持水合而在玻璃盖玻片最小化的XY偏移。用PBS施用的加热室的组合保持水合和动物的生理温度。这些条件组合保持血管灌注和血流以允许在肿瘤输精管细胞事件的扩展成像culature包括血管通透性和肿瘤细胞血管内。 这种技术的一个限制是可以用于可视化的细胞在肿瘤微环境的荧光标记的数目。产生转基因小鼠用含有荧光蛋白额外的细胞类型（ 即 ，内皮细胞，成纤维细胞或周细胞）可能是困难且耗时的。然而，未来的潜在应用可以利用与植入肿瘤细胞和巨噬细胞的70 kDa的葡聚糖标记基质的细胞类型的荧光蛋白标记的组合。通过使用这些标记的策略的组合，并且跨越转基因小鼠能够产生细胞标记的新组合，以评估在肿瘤微环境的细胞运动和事件。 不像基于免疫荧光染色终点实验，高分辨率活体显微镜允许的自发的和短暂的可视化事件在肿瘤微环境。此处描述的活体成像过程揭示了新的事件，在肿瘤微环境的细胞的相互作用，不能从静态图像解密。与上相互作用的细胞类型或过程，以及这些事件的动力学新颖的信息，可以生成假设来调查驱动这些相互作​​用的信号通路。

从初级乳腺肿瘤的肿瘤细胞的传播已经显示出不仅涉及肿瘤细胞，但承载间质细胞包括巨噬细胞和内皮细胞。此外，肿瘤血管异常与通透性增加1。因此，确定如何肿瘤细胞，巨噬细胞和内皮细胞相互作用介导的原发肿瘤微环境的血管通透性和肿瘤细胞血管内为理解转移重要。理解血管通透性，肿瘤细胞血管内和在肿瘤微环境的多细胞相互作用的基本信令机制的动力学可提供在抗癌疗法的开发和管理的关键信息。 研究体内肿瘤血管通透性的主要手段一直是血管外染料的测量，如伊文思蓝2，高分子量的葡聚糖（155 kDa）的染料注射后3和荧光团或放射性缀合的蛋白质（包括白蛋白）4在固定的时间点。在显微镜的进步，动物模型和荧光染料已通过活体显微镜5启用细胞过程和在活体动物血管通透性的可视化。 活的动物成像采集静态图像，或短的时间推移的序列在数个时间点不允许在肿瘤微环境6,7-动态事件的完整的理解。实际上，静态图像采集在24小时的过程中显示，肿瘤血管是漏水，但血管通透性的动力学没有观察到6。因此，延长的持续时间推移成像长达12小时捕获在肿瘤微环境动态事件的动力学。 本协议描述使用较长时间推移multiphot的关于活体显微镜来研究在肿瘤微环境动态多细胞事件。在肿瘤微环境多种细胞类型标记有可注射的染料或通过使用表达荧光蛋白的转基因动物。使用尾静脉导管的血管的染料或蛋白质可成像开始之后被注入以获得在肿瘤微环境的多细胞事件的动力学数据。活细胞成像的乳腺肿瘤是通过手术准备一个皮瓣露出。图像被获取用于使用配备有多个光电倍增管（PMT）探测器8多光子显微镜长达12小时。通过使用适当的过滤器，减法算法使4 PMT检测器获取在肿瘤微环境同时9 5的荧光信号。高分辨率多光子活体显微镜捕获在肿瘤微环境肿瘤 - 基质相互作用的单细胞分辨率成像，从而导致更好ü血管通透性和肿瘤细胞血管内10-13的nderstanding。具体地，扩展的活体成像显露高度局部，瞬态血管通透性，在肿瘤细胞，巨噬细胞和内皮细胞之间的相互作用的位点选择性地发生的事件（定义为转移，TMEM 14的肿瘤微环境）13。此外，肿瘤细胞血管内仅在TMEM发生和与血管通透性13被空间和时间上相关。这些事件的动态单细胞分辨率是通过在肿瘤微环境中使用荧光标记的细胞的延长的时间推移多光子显微镜的成为可能。

<table><tbody><tr><td>155 kDa dextran-tetramethylrhodamine isothiocyanate</td><td>Sigma Aldrich</td><td>T1287</td><td>reconstitute at 20 mg/ml in 1x PBS</td></tr><tr><td>70 kDa dextran-Texas Red</td><td>Life Technologies</td><td>D-1830</td><td>reconstitute at 10 mg/ml in 1x PBS</td></tr><tr><td>10 kDa dextran-fluorescein isothyocyanate</td><td>Sigma Aldrich</td><td>FD10S</td><td>reconstitute at 20 mg/ml in 1x PBS</td></tr><tr><td>Qdot 705 ITK Amino (PEG) Quantum Dots</td><td>Life Technologies</td><td>Q21561MP</td><td>Dilute 25 &amp;#956;l in 175 &amp;#956;l of 1x PBS for injection</td></tr><tr><td>MMTV-PyMT mice</td><td>Jackson Laboratory</td><td>2374</td><td></td></tr><tr><td>Csf1r-ECFP mice (Csf1r-Gal4/VP16,UAS-ECFP)</td><td>Jackson Laboratory</td><td>26051</td><td></td></tr><tr><td>Csf1r-EGFP mice</td><td>Jackson Laboratory</td><td>18549</td><td></td></tr><tr><td>1x PBS</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Isoethesia (isoflurane)</td><td>Henry Schein Animal Health</td><td>50033</td><td>250 ml</td></tr><tr><td>Oxygen</td><td>AirTech</td><td></td><td></td></tr><tr><td>1 ml syringe, tuberculin slip tip</td><td>BD</td><td>309659</td><td></td></tr><tr><td>30 G x 1 (0.3 mm x 25&amp;nbsp;mm) needle</td><td>BD</td><td>305128</td><td></td></tr><tr><td>Polyethylene micro medical tubing&amp;nbsp;</td><td>Scientific Commodities Inc</td><td>BB31695-PE/1</td><td>0.28 mm I.D. x 0.64 mm O.D.</td></tr><tr><td>Microscope coverglass</td><td>Corning</td><td>2980-225</td><td>thickness 1.5, 22 x 50&amp;nbsp;mm</td></tr><tr><td>PhysioSuite MouseSTAT pulse oximeter, software and sensors</td><td>Kent Scientific</td><td></td><td></td></tr><tr><td>Laboratory tape</td><td>Fisher Scientific</td><td>159015R</td><td></td></tr><tr><td>soft rubber pad</td><td>McMaster-Carr</td><td>8514K62</td><td>Ultra-Soft Polyurethane Film, 3/16&amp;rdquo; Thick, 12&quot; x 12&quot;, 40 Oo Durometer, Plain Back</td></tr><tr><td>hard rubber pad</td><td>McMaster-Carr</td><td>8568K615</td><td>High-Strength Neoprene Rubber Sheet 1/4&quot; Thick, 12&quot; x 12&quot;, 50&amp;nbsp;A Durometer</td></tr><tr><td>Microscope</td><td>Olympus</td><td></td><td>The microscope is a custom built two laser multiphoton microscope based on an Olympus IX-71 stand utilizing a 20X 1.05NA objective lens.</td></tr><tr><td>7-Punch set</td><td>McMaster-Carr</td><td>3429A12</td><td>1/4&quot; to 1&quot; Hole Diameter, for Hammer-Driven Hole Punch</td></tr></tbody></table>

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.

延长的时间推移活体显微镜使在肿瘤微环境的多细胞过程的单细胞分辨率的成像。通过荧光标记的肿瘤细胞，巨噬细胞，血管空间和可视化使用二次谐波生成信号的胶原纤维网络，在肿瘤微环境多个隔间成像期间被同时跟踪。标记的荧光蛋白的肿瘤细胞可以在转基因小鼠如已在MMTV-PyMT-Dendra2小鼠已经完成，用标记与Dendra2（ 图1A）的乳腺肿瘤细胞中产生。通过穿越CSF1R -GAL4VP16 / UAS-ECFP小鼠这些转基因小鼠，其具有标记的ECFP巨噬细胞，这两种肿瘤细胞和巨噬细胞被标记在MMTV-PyMT小鼠。与肿瘤细胞和巨噬细胞的双重标记策略，这两种细胞类型的直接相互作用可实时（ 图1A可视）。此外，用GFP转导的人乳腺癌细胞系或来源于患者的肿瘤细胞可被用于标记的肿瘤细胞。巨噬细胞可使用荧光标记的70 kDa的葡聚糖凝聚的吞噬的巨噬细胞（ 图1B）来标记。这些方法都促进了动态多的事件，如肿瘤细胞 - 巨噬细胞活力的流，血管通透性和肿瘤细胞血管内的研究。为了显现肿瘤血管通透性的改变，荧光标记的高分子葡聚糖（155 100kDa或更高推荐9,13,21,22）或量子点被用于标记血管空间（ 图1）。 155 kDa的葡聚糖或量子点是足够大，以致它们不容易通过interendothelial空间23扩散。 连续的高分辨率成像促进血管通透性事件和捕获右旋糖酐外渗和间隙动力学在量化TMEM（ 图2和电影1）。由于血管间隙很容易通过155 kDa的葡聚糖在时间推移序列的开始（ 图2的在0&#39;）定义的血管外荧光葡聚糖所用的时间推移序列的每个帧进行量化。 图2和电影1展示外渗155 kDa的葡聚糖TMR通过尾静脉导管插入MMTV-PyMT-Dendra2注射; CSF1R-CFP鼠标。 Dendra2标记的肿瘤细胞（绿色）和CFP标记的巨噬细胞（青色）用于在活的动物，以确定TMEM。在血管通透性的部位TMEM结构在白箱中概述。血管内标有155 kDa的葡聚糖TMR（红色）可视化血管通透性的事件中流动的血管和血管内容外渗。 155 kDa的葡聚糖TMR外渗的峰值在图2中看到的29&#39;和34&#39;之间。血管外葡聚糖位在白箭头在顶部板和在该下面板的红色箭头，仅显示155 kDa的葡聚糖的TMR的信道。的155 kDa的葡聚糖的TMR从组织清除和被视为在如在图2的97&#39;看到的血管外的TMR信号的下降。在ImageJ的血管外葡聚糖时延序列的汇编量化后。 除了在肿瘤微环境观察自发事件，它来研究的识别现象的潜在的分子机制是重要的。注入额外血管染料或蛋白质以诱导血管通透性能提供深入了解调节血管渗透性的信号传导事件。 图3展示了在10 kDa的葡聚糖-FITC（绿色）和155 kDa的葡聚糖TMR（红色）的外渗的差。超高分子量155 kDa的葡聚糖TMR是时间推移成像开始前立即服用。五的Z堆叠在时间推移序列获得设置一个基线注入10 kDa的葡聚糖-FITC之前。采集的第 5 次后的Z-堆叠10 kDa的葡聚糖-FITC是通过尾静脉导管施用，而不中断图像采集。葡聚糖-FITC看出进入即时外渗脉管而155 kDa的葡聚糖的TMR仍然局限在血管空间（ 图3,0&#39;和6&#39;）。 10 kDa的葡聚糖-FITC从血管完全extravasates和从离开155 kDa的葡聚糖TMR在血管（ 图3中在56&#39;和电影2所见）组织清除。从使用注射前的时间点，10 kDa的葡聚糖-FITC外渗的动力学可以很容易地比较，155 kDa的葡聚糖TMR 13。 10 kDa的dextr的直接外渗注射后-FITC是从155 kDa的葡聚糖的TMR在脉管保留和自发血管通透性的事件13显著不同。附加的实验控制可以包括通过内皮或通过VEGFA 165介导的渗透性13的机械破坏诱发血管通透性来执行。自发渗透性事件155 kDa的葡聚糖的TMR外渗的动力学可以比的10kDa葡聚糖，和诱导血管通透性理解涉及在肿瘤微环境的血管通透性的事件的事件和信号的其它手段。 <img alt="图1" src="/files/ftp_upload/54042/54042fig1.jpg" /> 图1.策略在肿瘤微环境标记细胞和血管。（A）中的转基因MMTV-PyMT小鼠用标记与Dendra2肿瘤细胞（MMTV-ICRE / CAGCAT-Dendra2 / MMTV-PyMT），并标有CFP巨噬细胞（CSF1R -GAL4VP16 / UAS-ECFP）。肿瘤血管标有由尾静脉导管给予155 kDa的葡聚糖TMR。 （B）的用标记70 kDa的葡聚糖德克萨斯红巨噬细胞和血管标记使用尾静脉导管量子点TN1-GFP患者来源异种移植肿瘤。比例尺，50微米。 <a href="https://www.jove.com/files/ftp_upload/54042/54042fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54042/54042fig2.jpg" /> 图2.可视化的瞬态血管通透性。（A）活体显微镜（IVM）155 kDa的葡聚糖TMR（红色）外渗和肿瘤细胞血管内的在TMEM时间推移（白盒）。标有Dendra2（绿色，TC）与巨噬细胞ECFP（青色，M）和一个BLO肿瘤细胞OD艘，155 kDa的葡聚糖TMR（红，BV）。血管通透性网站（白色箭头）。 （B）隔离155 kDa的葡聚糖TMR通道。红色箭头标记右旋糖酐外渗（白色）。比例尺，50微米。在电影1时间推移显微镜的电影。 <a href="https://www.jove.com/files/ftp_upload/54042/54042fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54042/54042fig3.jpg" /> 图3.可视多种荧光标记的右旋糖苷。在155 kDa的葡聚糖TMR（红色）和10kDa葡聚糖-FITC（绿色）的外渗差的IVM时间推移。巨噬细胞标记ECFP（蓝绿色）和蓝色（SHG）的胶原蛋白。红色155 kDa的葡聚糖TMR在血管的覆盖和绿色10 kDa的葡聚糖-FITC是黄色的。绿色10 kDa的葡聚糖-FITC峰迅速外渗是6分钟。 SCALË酒吧，50微米。在电影2时间推移显微镜的电影。 <a href="https://www.jove.com/files/ftp_upload/54042/54042fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="电影1" src="/files/ftp_upload/54042/54042movie1.jpg" /> 电影1.可视自发瞬时血管通透性随时间推移IVM。定时显微镜（如见图2）在转基因MMTV-PyMT小鼠用标记与Dendra2（MMTV-ICRE / CAGCAT肿瘤细胞可视化的瞬态血管通透性的-Dendra2 / MMTV-PyMT），并标有CFP（CSF1R -GAL4VP16 / UAS-ECFP）的巨噬细胞。 155 kDa的葡聚糖TMR外渗（红在左侧面板，白色右图）外渗在肿瘤血管的分支点观察。 <a href="https://www.jove.com/files/ftp_upload/54042/Movie_1-JoVE_Harney.avi" target="_blank">P</a>租赁点击这里观看该视频。 （右键点击下载）。 <img alt="电影2" src="/files/ftp_upload/54042/54042movie2.jpg" /> 电影2.时间推移，IVM可视多种荧光标记的葡聚糖的外渗。在转基因155 kDa的葡聚糖TMR（红色）和10kDa葡聚糖-FITC（绿色）的外渗的定时显微镜（如见于图3） MMTV-PyMT鼠标标有CFP（CSF1R -GAL4VP16 / UAS-ECFP）的巨噬细胞。胶原SHG被标记为蓝色。红155 kDa的葡聚糖TMR保持在肿瘤血管的时间推移的持续时间，而绿10 kDa的葡聚糖-FITC迅速extravasates从血管和组织清除。 <a href="https://www.jove.com/files/ftp_upload/54042/Movie_2-JoVE_Harney.avi" target="_blank">请点击此处观看该视频。 （右键点击下载）。</a>

Watch this Scientific Journal Video about Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment at JoVE.com

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

这个协议描述了使用多光子显微术的实时在单细胞分辨率来执行多细胞相互作用的延长时间推移成像，体内 。

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 ...

extended-time-lapse-intravital-imaging-real-time-multicellular

Nanyang Technological University

Research

JoVE Journal

Medicine

8.1K 次观看.  Albert Einstein College of Medicine. 这个协议描述了使用多光子显微术的实时在单细胞分辨率来执行多细胞相互作用的延长时间推移成像，体内 。 

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

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung is the most difficult to assess using intravital optical imaging due to its enclosed position within the body, delicate nature and vital role in sustaining proper physiology. While clinical modalities (positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT)) are capable of providing noninvasive images of this tissue, they lack the resolution necessary to visualize the earliest seeding events, with a single pixel consisting of nearly a thousand cells. Current models of metastatic lung seeding postulate that events just after a tumor cell&#39;s arrival are deterministic for survival and subsequent growth. This means that real-time intravital imaging tools with single cell resolution 2 are required in order to define the phenotypes of the seeding cells and test these models. While high resolution optical imaging of the lung has been performed using various ex vivo preparations, these experiments are typically single time-point assays and are susceptible to artifacts and possible erroneous conclusions due to the dramatically altered environment (temperature, profusion, cytokines, etc.) resulting from removal from the chest cavity and circulatory system 3. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window 2,4,5 however, typical imaging times have been limited to approximately 6 hr. Here we describe a protocol for performing long-term intravital time-lapse imaging of the lung utilizing such a window over a period of 12 hr. The time-lapse image sequences obtained using this method enable visualization and quantitation of cell-cell interactions, membrane dynamics and vascular perfusion in the lung. We further describe an image processing technique that gives an unprecedentedly clear view of the lung microvasculature.

This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

长期高分辨率活体显微镜在肺与真空稳定成像窗口

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung ...

科研

癌症研究

Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized treatment determination. However, perturbation of the tumor architecture by biopsy and other invasive procedures has been associated with undesired effects on tumor progression, which need to be studied in depth to further improve the clinical benefit of these procedures. Conventional static approaches, which only provide a snapshot of the tumor, are limited in their ability to reveal the impact of biopsy on tumor cell behavior such as migration, a process closely related to tumor malignancy. In particular, tumor cell migration is the key in highly aggressive brain tumors, where local tumor dissemination makes total tumor resection virtually impossible. The development of multiphoton imaging and chronic imaging windows allows scientists to study this dynamic process in living animals over time. Here, we describe a method for the high-resolution longitudinal imaging of brain tumor cells before and after a biopsy in the same living animal. This approach makes it possible to study the impact of this procedure on tumor cell behavior (migration, invasion, and proliferation). Furthermore, we discuss the advantages and limitations of this technique, as well as the ability of this methodology to study changes in the cancer cell behavior for other surgical interventions, including tumor resection or the implantation of chemotherapy wafers.

Here we describe a method for high-resolution time-lapse multiphoton imaging of brain tumor cells before and after invasive surgical intervention (e.g., biopsy) within the same living animal. This method allows studying the impact of these invasive surgical procedures on tumor cells&#39; migratory, invasive, and proliferative behavior at a single cell level.

脑肿瘤细胞行为的纵向生命成像,以响应侵入性手术活检

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized ...

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 ...

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

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

与落射荧光组织基质成分和双光子显微镜的长期活体免疫荧光成像

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

生物工程

A Permanent Window for Investigating Cancer Metastasis to the Lung

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as the bone, brain, and lung. Although extensively studied, the mechanistic details of this process remain poorly understood. While common imaging modalities, including computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging (MRI), offer varying degrees of gross visualization, each lacks the temporal and spatial resolution necessary to detect the dynamics of individual tumor cells. To address this, numerous techniques have been described for intravital imaging of common metastatic sites. Of these sites, the lung has proven especially challenging to access for intravital imaging owing to its delicacy and critical role in sustaining life. Although several approaches have previously been described for single-cell intravital imaging of the intact lung, all involve highly invasive and terminal procedures, limiting the maximum possible imaging duration to 6-12 h. Described here is an improved technique for the permanent implantation of a minimally invasive thoracic optical Window for High-Resolution Imaging of the Lung (WHRIL). Combined with an adapted approach to microcartography, the innovative optical window facilitates serial intravital imaging of the intact lung at single-cell resolution across multiple imaging sessions and spanning multiple weeks. Given the unprecedented duration of time over which imaging data can be gathered, the WHRIL can facilitate the accelerated discovery of the dynamic mechanisms underlying metastatic progression and numerous additional biologic processes within the lung.

Here, we present a protocol for the surgical implantation of a permanently indwelling optical window for the murine thorax, which enables high-resolution, intravital imaging of the lung. The permanence of the window makes it well-suited to the study of dynamic cellular processes in the lung, especially those that are slowly evolving, such as metastatic progression of disseminated tumor cells.

研究癌症转移至肺部的永久窗口

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as ...

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse images of mammary tumor models to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable.

The intravital imaging method described here utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for in-depth analysis of 4D intravital images.

一种用于乳腺肿瘤微环境活体内成像的无标记分割方法

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

在髓系细胞动态实时成像<em&gt;装甲运兵车<sup&gt;最小/ +</sup</em&gt;肠道肿瘤通过旋转盘共聚焦显微镜

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

免疫与感染

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment.

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

We describe a relatively simple method for ex vivo live imaging of the tumor cell-stroma interactions within lung metastasis, utilizing fluorescent reporters in mice. Using spinning-disk confocal microscopy, this technique enables visualization of live cells for at least 4 hr and could be adapted to study other inflammatory lung conditions.

体外肺转移及微环境的实时成像

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular ...

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.

实时多细胞动力学延长时间推移活体成像在肿瘤微环境

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