Name: ex vivo live imaging of lung metastasis and their microenvironment
Uploaded: 2016-02-03T14:00:00.000+00:00
Duration: 11 min 19 s
Description: Watch this Scientific Journal Video about Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment at JoVE.com

We thank Nguyen H. Nguyen for her technical help and Audrey O&#8217;Neill for support with the Zeiss Cell Observer spinning-disk confocal microscope. This work was supported by a Department of Defense postdoctoral fellowship (W81XWH-11-01-0139) and the Weizmann Institute of Science-National Postdoctoral Award Program for Advancing Women in Science (to V.P.).

中描述的所有程序必须按照与使用脊椎动物，包括由当地机构动物护理和使用委员会（IACUC）批准的准则和规定进行。 1.产生肺转移瘤防爆体内实时成像（转基因或尾静脉注射） 注:肺转移可通过利用遗传工程小鼠模型或通过静脉内（IV）注射癌细胞产生。 <ol><li>通过杂交的基因工程肿瘤小鼠模型成转基因记者鼠标，例如，生成用于成像肺转移，穿过乳腺癌小鼠模型中，小鼠乳腺肿瘤病毒长末端重复序列，多瘤中间T抗原（MMTV-PyMT）11到ACTB-ECFP小鼠模型12。 注:ACTB-ECFP模型表达β幕下增强青色荧光蛋白（ECFP）在启动使得所有的细胞发出荧光的蓝色，CFP通道。然而，肿瘤细胞是迄今为止最突出并显示为在显微镜下的堆积ECFP阳性细胞。的MMTV-PyMT小鼠模型开发一种进行性疾病，其乳腺肿瘤的生长与癌细胞传播到外围相关，特别是到肺部。在上FVB / N背景MMTV-PyMT小鼠中，微可以约10-11周龄被观察到。一般来说，这些进步macrometastases在14周13岁 。 要么</li><li>生成使用原代细胞或同基因细胞系的实验转移。使用在随后静脉注射14 体外操纵原发肿瘤细胞或细胞系（例如，转导）。 <ol><li>简要地说，在这个协议中，注入一个绿色荧光蛋白（GFP）-expressing（+），MMTV-PyMT细胞系成荧光报道小鼠（ACTB-ECFP）或野生型小鼠。然后，可视化使用绿色，GFP通道，这些细胞被称为VO-PyMT细胞15。 注:原VO-PyMT细胞系在骨科范德比尔特纳什维尔的。 VO表示范德比尔特骨科。 </li><li>以下10 6个细胞的注射（200微升），立即向上观察癌细胞外渗到注射后的几个小时;注射后观察1-3周之间的微小转移灶和注射后15检测macrometastases 3周。 注:较少的细胞可以被注射到延长从注射到转移生长的时间。 </li></ol></li></ol> 2.标签在转移性微环境利益成分（转基因和/或注射剂） 注意:标记可以通过转基因小鼠和/或通过各种注射来实现。确保使用不同的荧光色为各种细胞类型的标签。 <ol><li>使用转基因小鼠的转移性微环境的标签组件。越过前面提到的小鼠肿瘤模型（例如，MMTV-PyMT点¯xACTB-ECFP）插入，其中所关注的基质细胞通过荧光蛋白，其是不ECFP，例如，C-fms的-EGFP标记的转基因小鼠模型4,16。 注:除癌细胞在CFP通道的可视化，这使骨髓细胞的可视化在GFP 通道 4。 AND / OR </li><li>标签使用注射到转基因荧光报告小鼠或（非荧光）的野生型小鼠中的转移性微环境的各种组件。 注意:一些化合物可以注射来标记转移性微环境，例如，一个AF647共轭Gr-1抗体在这里用于标记的中性粒细胞和单核细胞的一些13和不同分子量右旋糖酐使用的各种部件标签肺毛细血管。用于制备这些注射参见步骤4。 </li></ol> 3.材料的制备解剖前<ol><li> 2％琼脂糖<ol><li>称取0.2克琼脂糖，并添加至10ml 1×PBS。加热该溶液以溶解琼脂糖。琼脂糖将巩固在室温，因此它保持在37℃水浴直至用于通货膨胀。 </li></ol></li><li> 的 CO 2和温度控制器<ol><li>检查的DDH 2 O在加湿室。在需要的时候笔芯。将配置板进入下阶段的板夹（气候箱）。转动的 CO 2控制器上，并在5％的设定的 CO 2。确保气流速率设定为0.4升/分钟。 </li><li>打开空气和CO 2阀。转温度控制器上。确保气候室的温度和盖都设置在37℃。 </li><li>释放的 CO 2米气压力。检查二氧化碳增加，Equilibration可能需要长达30分钟。 </li></ol></li><li>旋转盘共聚焦显微镜 注:在显微镜设置的细节先前已经描述4,17。 <ol><li>开启激光器（氩激光为488纳米激发和固态405纳米，561纳米和640纳米激光器）。打开显微镜，照相机，纺丝盘控制单元，AOTF中，激光控制单元和摄像机控制器。 </li><li>打开显微镜快门，打开电脑上运行的显微镜和打开软件。 </li></ol></li><li>工具和平台解剖准备。 <ol><li>打开热珠灭菌，让它达到250℃。清洁2对手术剪刀和镊子用水和肥皂。消毒的工具为至少30秒。让工具冷却。使用聚苯乙烯作为盖夹层平台。用一块实验室透雨的覆盖。 </li></ol></li></ol> 4.注射的制备 注意:根据半衰期，优选的响应，注射荧光标记的抗体和/或荧光分子或之前立即处死动物或前几个小时到几天。 <ol><li>到图像Gr1的阳性嗜中性粒细胞和单核细胞，制备具有库存AF647共轭Gr-1抗体的7微升（1毫克/毫升）到罩下加入100μl无菌PBS的注射器。广场上的注射器27 G½针头。 </li><li>到图像肺毛细血管，准备用100μl任一70 kD的若丹明共轭葡聚糖（4毫克/毫升）或10 kD的AF647共轭葡聚糖（4毫克/毫升）的第二和第三注射器。广场上的注射器27 G½针头。 </li><li>注射前，肺部的切除的AF647标记的抗体溶液IV 5小时。 </li><li>注入四之前立即肺部的切除一个或两个葡聚糖溶液。 </li></ol> 5.准备肺部前体内实时成像 注:尽量上班无菌和小心，尽量避免肺部内的免疫细胞的不必要的挑战。 <ol><li>与由IACUC批准的动物协议， 如:允许麻醉致死过量注入小鼠腹膜内（IP），将1ml 2.5％阿佛丁的。等待鼠标停止呼吸，完全不响应伤害性刺激（后肢捏）。 注:颈椎脱位和二氧化碳安乐死应该避免，因为它可以不利地影响肺细胞生存力。 </li><li>固定在清扫板鼠标和消毒用70％乙醇鼠标。 </li><li>用手术剪先使通过皮肤的横向胃脘切口，然后通过腹膜类似的切口。保持夹层板在垂直位置并切断降主动脉，使血液池向下腹部而不是在胸腔。 </li><li>小号消灭在膜片释放真空的小开口。沿10和第12肋骨切切除膈肌，并获得了肺部视觉访问。 </li><li>用手术剪刀切开皮肤至气管在胸腔，但留下胸腔完好。独立于胸腔皮肤。通过去除周围结缔组织，注意不要损伤气管本身（ 图1A）暴露气管。 </li><li>剪断的小开口在平行于软骨环暴露气管直径约为1毫米，如靠近喉部尽可能（图1B）。要小心，不要通过气管完全切断。 </li><li>取一个20G的针和轻轻插入针4-5毫米进气管没有任何反力（图1D）。针的端部应通过气管（图1C）可见。使用镊子稳定气管针。或者，缝线可以阿罗绑UND气管以保持针就位。 注:通过插入过深，隆突可能会受到创伤，或只有一个肺的一侧可能被夸大。 </li><li>填的注射器用400μl37℃的2％低熔点温度琼脂糖（直接从恒温浴中取出）的。确保夹层板站起来，慢慢地灌输通过针进入肺内温暖琼脂糖，使用〜400微升膨胀肺部。 注:观看肺部胸腔内充气。不要过分夸大，因为它会破裂的肺。 </li><li>一旦肺充气，充〜胸廓的⅔，拆下注射器和保持气管内的针，以防止任何琼脂糖泄漏。 </li><li>倒在充气肺部大约50ml 20℃的PBS，以允许肺部内的琼脂糖设置和固化。慢慢用镊子取出针并关闭气管，以防止任何非固化琼脂糖泄漏。&lt;/ LI&gt; <li>通过执行胸骨切开术暴露肺部并随后切除肺。对于肺切除，保住气管，同时通过气管切割彻底。轻轻拉动气管上，切去结缔组织和食道同时拉动肺出胸腔直到肺部从小鼠（图1E）分离。 </li><li>沉浸在温暖的RPMI-1640肺部洗掉过量的血液，并通过使用剪刀和镊子切断裂片&#39;主茎支气管在脐（图1F）的裂片轻轻分开。 </li><li>放置裂片，与扁平表面向下最大化成像表面，在24孔成像板（图1G）的孔中。添加在叶前100微升37℃RPMI-1640。放置在叶片的顶部几15毫米圆形显微镜盖玻片以防止其浮动。 </li><li>倾温PBS到周围井，以防止从EVAP所述的RPMI-1640培养基orating。插入24孔板进平衡气候室并保持在37℃的肺叶与空气和5％的CO 2。插入气候室上的共焦显微镜的阶段。 注意:其他气体的混合物（例如，5％O 2，5％CO 2的N 2以检查缺氧/低氧气的条件下，细胞的行为）也可以考虑。 </li></ol><img alt="图1" src="/files/ftp_upload/53741/53741fig1.jpg" /> 图1.协议的准备肺部实时成像的。（A）准备鼠标后气管暴露。在平行于软骨环暴露气管制成（B）的小剪断。 （C），20G的针头插入4-5毫米的进气管。 （D）的400微升的2％低熔点温度琼脂糖到肺部的灌输。 （E）INFLated肺部鼠标分开。充气后（F）裂片分离。 （G）叶放置在一个24孔成像板的好。 <a href="https://www.jove.com/files/ftp_upload/53741/53741fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 6.采集和图像分析 注:图像可以与各种纺丝通过各种软件程序支持的磁盘共焦显微镜的获取。在这个协议中，无论是与一个特制的旋转盘共聚焦显微镜或禅与市售的旋转盘聚焦显微镜μManager用于图像采集，而了Imaris用于电影编辑和分析。 <ol><li>获取使用μManager图像。收购使用μManager软件映像的详细分步协议是前面所述18。 要么</li><li>获取图像使用图像分析软件，如禅（ 见图S1）。 <ol><li>点击"查找"选项卡，并选择在"光路" 工具 （图S1A，红色框）目标（10倍20倍或）。随后，点击"眼睛- DAPI&#39;看看通过目镜（图S1A，蓝盒）CFP通道。本地化样品使用显微镜手动。点击"所有Off&#39;after组织是视野的中心。 </li><li>点击"捕获"选项卡，设置图像采集的所有参数。 </li><li>在"渠道"工具，单击"+"按钮（图S1B，红色框）。一个弹出菜单显示和搜索染料（S）目前在"染料数据库"（图S1B）的样本。选择染料，然后单击"添加"。 注:该计划将设置为优化所有过滤器。染料可以删除通过选择依次点击垃圾桶按钮 （图S1B，黄框）。 </li><li>在"采集模式"菜单中，将"像素合并"到5x5的。在渠道上菜单上ECFP双击将其选中。降低激光功率到20％，使样品不会同时进行图像采集设置的参数进行漂白。 </li><li>选中"瓷砖"框在"实验管理"部分，瓷砖工具出现在"多维收购"工具组（ 图S1C）英寸点击"高级设置"按钮，从摄像机查看实时图像。在"位置"部分，点击"添加"按钮，4〜6个位置添加到实验。要删除的位置，选择位置，单击垃圾箱按钮。 </li><li>在"采集参数"工具组中，打开了"聚焦战略"工具，然后选择"绝对E固定从下拉列表中Z位置"。 </li><li>检查的Z-Stack框在"实验管理"部分和的Z-Stack工具出现在"多维收购"工具组（ 图S1D）英寸上的一个位置双击在"位置"部分，然后按&#39;活&#39;。手动先设置和设置的摄像视场的最后位置。于4微米的设置间隔。 注意:程序将确定切片为选定的范围和时间间隔的数目。理想的情况下，5-7片方便，以便有足够的可视化和快速的图像采集。 </li><li>检查在"实验管理"部分的"时间序列"对话框。设置所需的"时间"和"间隔"中的"时间序列"的工具，出现在"多维收购"工具组（ 图S1E）的时间。 </li><li>在&#39;习得化模式"菜单中，将"像素合并"2×2。在通道菜单中荧光双击选中它并增加激光功率为100％。按"现场"，并调整"曝光时间"。重复此为每个荧光基团。 </li><li>选中"启用自动保存"复选框。在文件名选择一个文件夹和类型。所有采集的图像会自动保存此文件夹中。 </li><li>在"实验管理"部分，点击"开始试验"开始图像采集。 </li></ol></li><li>图像采集后，编译了Imaris软件的原始数据。将图像转换为.ims文件，并可以进行调节。对于文件的转换，进行调整，并使用了Imaris保存电影的详细分步协议是前面所述18。 </li><li>当保存影片，设定每秒（fps）的"帧率"5张。 </li></ol>

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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+application+of+a+murine+bone+bioreactor+as+a+model+of+tumor:+bone+interaction.">The application of a murine bone bioreactor as a model of tumor: bone interaction.</a> Clin Exp Metastas. 23 (7-8), 345-356 (2006).</li><li>Sasmono, R. T., Oceandy, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+macrophage+colony-stimulating+factor+receptor-green+fluorescent+protein+transgene+is+expressed+throughout+the+mononuclear+phagocyte+system+of+the+mouse.">A macrophage colony-stimulating factor receptor-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse.</a> Blood. 101 (3), 1155-1163 (2003).</li><li>Ewald, A. J., Werb, Z., Egeblad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic,+long-term+in+vivo+imaging+of+tumor-stroma+interactions+in+mouse+models+of+breast+cancer+using+spinning-disk+confocal+microscopy.">Dynamic, long-term in vivo imaging of tumor-stroma interactions in mouse models of breast cancer using spinning-disk confocal microscopy.</a> Cold Spring Harb Protoc. (2), (2011).</li><li>Bonnans, C., Lohela, M., Werb, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+of+myeloid+cells+dynamics+in+ApcMin/++intestinal+tumors+by+spinning+disk+confocal+microscopy.">Real-time imaging of myeloid cells dynamics in ApcMin/+ intestinal tumors by spinning disk confocal microscopy.</a> J Vis Exp. (92), (2014).</li><li>Nakasone, E. S., Askautrud, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+tumor-stroma+interactions+during+chemotherapy+reveals+contributions+of+the+microenvironment+to+resistance.">Imaging tumor-stroma interactions during chemotherapy reveals contributions of the microenvironment to resistance.</a> Cancer cell. 21 (4), (2012).</li><li>Cheng, N., Lambert, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammary+transplantation+of+stromal+cells+and+carcinoma+cells+in+C57BL/6J+mice.">Mammary transplantation of stromal cells and carcinoma cells in C57BL/6J mice.</a> J Vis Exp. (54), (2011).</li><li>Al-Mehdi, A. B., Tozawa, K., Fisher, A. B., Shientag, L., Lee, A., Muschel, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+origin+of+metastasis+from+the+proliferation+of+endothelium-attached+tumor+cells:+a+new+model+for+metastasis.">Intravascular origin of metastasis from the proliferation of endothelium-attached tumor cells: a new model for metastasis.</a> Nat Med. 6 (1), 100-102 (2000).</li><li>Wong, C. W., Song, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+location+of+breast+cancer+cells+after+spontaneous+metastasis+to+the+lung.">Intravascular location of breast cancer cells after spontaneous metastasis to the lung.</a> Am J Pathol. 161 (3), 749-753 (2002).</li><li>Liang, C. -. C., Park, A. Y., Guan, J. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+scratch+assay:+a+convenient+and+inexpensive+method+for+analysis+of+cell+migration+in+vitro.">In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro.</a> Nat protoc. 2 (2), 329-333 (2007).</li><li>Nelson, K., Bobba, C., Ghadiali, S., Hayes, D. J., Black, S. M., Whitson, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+ex+vivo+lung+perfusion+as+a+platform+for+transplantation+research.">Animal models of ex vivo lung perfusion as a platform for transplantation research.</a> World J Exp Med. 4 (2), (2014).</li><li>Magness, S. T., Bataller, R., Yang, L., Brenner, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dual+reporter+gene+transgenic+mouse+demonstrates+heterogeneity+in+hepatic+fibrogenic+cell+populations.">A dual reporter gene transgenic mouse demonstrates heterogeneity in hepatic fibrogenic cell populations.</a> Hepatology. 40 (5), 1151-1159 (2004).</li><li>Motoike, T., Loughna, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+GFP+reporter+for+the+study+of+vascular+development.">Universal GFP reporter for the study of vascular development.</a> Genesis. 28 (2), (2000).</li><li>Srivastava, M. K., Andersson, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid+suppressor+cells+and+immune+modulation+in+lung+cancer.">Myeloid suppressor cells and immune modulation in lung cancer.</a> Immunotherapy. 4 (3), (2012).</li><li>Craig, A., Mai, J., Cai, S., Jeyaseelan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+recruitment+to+the+lungs+during+bacterial+pneumonia.">Neutrophil recruitment to the lungs during bacterial pneumonia.</a> Infect Immun. 77 (2), 568-575 (2009).</li><li>Kreisel, D., Nava, R. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+two-photon+imaging+reveals+monocyte-dependent+neutrophil+extravasation+during+pulmonary+inflammation.">In vivo two-photon imaging reveals monocyte-dependent neutrophil extravasation during pulmonary inflammation.</a> Proc Natl Acad Sci USA. 107 (42), 18073-18078 (2010).</li></ol>

The authors have no conflicts of interest to disclose. All animal experiments were conducted in accordance with IACUC approved protocols, UCSF.

这份手稿描述了转移的小鼠模型体外肺转移的实时成像的详细方法。该成像协议提供了肺的微环境中的动态和空间肿瘤细胞 - 基质相互作用的直接可视化。它是一个相对容易的和快速的方法，它允许为至少4小时的肺转移的可靠的成像。从这些实验中获得的影可以用来跟踪动态过程如细胞运动和细胞相互作用。 被描述为肺转移的产生方法有两种:一种基因工程小鼠模型和...

The deadliest aspect of cancer is metastasis, which accounts for more than 90% of cancer-related morbidity and mortality1. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular mechanisms that govern metastatic dissemination and growth are still elusive. To metastasize, tumor cells in the primary tumor must detach from their neighboring cells and basement membrane, cross through the extracellular matrix, intravasate, travel via blood or lymphatic vessels, extravasate at the secondary site, and finally, survive and establish secondary tumors. In addition to the properties of the tumor cells, the contribution from the microenvironment, which includes the adjacent stroma along with the normal counterparts of the cancer cells, is crucial for the seeding and establishment of metastatic lesions2.
Traditional methods to study metastatic seeding and growth examine static states, as tissues are excised and sectioned for histology. These data only generate a snapshot of this highly dynamic process. Although some useful information can be gained from these studies, the complicated process by which tumor and stromal cells interact during metastatic formation cannot be adequately assessed by these methods. Furthermore, it is not possible to gain insights into tumor or stromal cell migration patterns, which are important in establishing a colony at the distant site. In order to effectively study the metastatic process, it is essential to visualize various interactions between cancer cells and their microenvironment in a continuous manner and at real time. 
The lung is a common site for metastases from solid tumors as breast, colorectal, pancreatic cancer, melanoma and sarcoma3. Intravital imaging was previously used to study cell-cell interaction in various primary tumor and metastatic models4,5. Methods of lung imaging in mice, including intravital imaging, lung section imaging, and an ex vivo pulmonary metastasis assay have been published6&#8211;9. Intravital imaging of mouse lungs utilizes a thoracic suction window to stabilize the lungs6. This method is used for time-lapse imaging of the lung microcirculation and alveolar spaces. The anatomical location of the lungs poses a challenge to intravital imaging. In order to access the lungs, the chest cavity must be opened which leads to loss of negative pressure and collapsed lungs. This method only allows the visualization of a small part of the lungs and is technically demanding; an unnecessary complication in studies that examine processes that are independent of blood flow. Moreover, this method also requires gating out movement caused by breathing. This is done either by collecting images between breaths or during post image acquisition analyses10. The alternative ex vivo lung section imaging provides stability and depth, and also prepares lung parenchyma for immunostaining7. However, the lengthy sectioning process leads to an extensive delay between the time of animal sacrifice and the start of the imaging session. Moreover, the process of sectioning a mouse lung causes considerable amount of cell death8, thus interfering with the quality and quantity of imaging samples and perhaps needlessly altering tumor-stroma interactions. In order to technically bridge between the methods of intravital imaging and lung section imaging, while exploiting the advantages of the two techniques, a relatively fast and easy method for ex vivo lung imaging was developed. This method was achieved by imaging of non-sectioned whole lung lobes. 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.

<table><tbody><tr><td>MMTV-PyMT/FVB mice</td><td>Jackson Laboratory</td><td>2374</td><td>Female mice</td></tr><tr><td>ACTB-ECFP/FVB mice</td><td>UCSF Werb lab</td><td></td><td>Female mice</td></tr><tr><td>c-fms-EGFP/FVB mice</td><td>UCSF Werb lab</td><td></td><td>Female mice</td></tr><tr><td>FVB mice</td><td>Jackson Laboratory</td><td>1800</td><td>Female mice</td></tr><tr><td>GFP+ VO-PyMT cells</td><td>UCSF Werb lab</td><td></td><td></td></tr><tr><td>70,000 kDa Dextran, rhodamine-conjugated</td><td>Invitrogen</td><td>D1818</td><td>Dilute to&amp;nbsp; 4mg/ml in 1 x PBS and store at -20&amp;nbsp; &amp;deg;C. Use 0.4 mg per animal.&amp;nbsp;</td></tr><tr><td>10,000 kDa Dextran, Alexa Fluor 647 conjugated</td><td>Invitrogen</td><td>D22914</td><td>Dilute to&amp;nbsp; 4mg/ml in 1 x PBS and store at -20&amp;nbsp; &amp;deg;C. Use 0.4 mg per animal.&amp;nbsp;</td></tr><tr><td>Anti-mouse Gr-1 antibody Alexa Fluor 647</td><td>UCSF Monoclonal antibody core</td><td></td><td>Stock 1mg/ml. Use 7 ug per animal.</td></tr><tr><td>Anesthetic</td><td></td><td></td><td>Anesthesia approved by IACUC, used for anesthesia and/or euthanesia</td></tr><tr><td>1X PBS</td><td>UCSF cell culture facility</td><td></td><td></td></tr><tr><td>PBS, USP sterile&amp;nbsp;</td><td>Amresco INC</td><td>K813-500ML</td><td>Ultra pure grade for i.v. injection</td></tr><tr><td>Styrofoam platform</td><td></td><td></td><td>Will be used as dissection board</td></tr><tr><td>Fine scissors sharp&amp;nbsp;</td><td>Fine Science Tools</td><td>14060-11</td><td></td></tr><tr><td>Forceps</td><td>Roboz Surgical Store</td><td>RS-5135</td><td></td></tr><tr><td>Hot bead sterilizer</td><td>Fine Science Tools</td><td>18000-45</td><td>Turn ON 30min before use</td></tr><tr><td>Air</td><td>UCSF</td><td></td><td></td></tr><tr><td>Oxygen</td><td>UCSF</td><td></td><td></td></tr><tr><td>Carbon dioxide</td><td>UCSF</td><td></td><td></td></tr><tr><td>1 mL syringe without needle&amp;nbsp;</td><td>BD</td><td>309659</td><td></td></tr><tr><td>27 G x 1/2 needle&amp;nbsp;&amp;nbsp;</td><td>BD</td><td>305109</td><td>for i.v. injection</td></tr><tr><td>20 G x 1 needle, short bevel&amp;nbsp;&amp;nbsp;</td><td>BD</td><td>305178</td><td></td></tr><tr><td>Low-melting-temperature agarose&amp;nbsp;</td><td>Lonza</td><td>50111</td><td>To make 10 ml of solution, weigh 0.2 g of agarose, add to 10 ml 1 x PBS, and heat to dissolve. Agarose will solidify at room temperature, so maintain in a 37 &amp;deg;C water bath until used for inflation.</td></tr><tr><td>RPMI-1640 medium without phenol red</td><td>Life Technologies</td><td>11835-030</td><td></td></tr><tr><td>24 well Imaging plate&amp;nbsp;</td><td>E&amp;amp;K scientific</td><td>EK-42892</td><td></td></tr><tr><td>Glass cover slides, 15 mm&amp;nbsp;</td><td>Fisher Scientific</td><td>22-031-144</td><td></td></tr><tr><td>Digital CO2 and temperature controller</td><td>Okolab</td><td>DGTCO2BX</td><td>http://www.oko-lab.com</td></tr><tr><td>Climate chamber</td><td>Okolab</td><td></td><td>http://www.oko-lab.com</td></tr><tr><td>Cell Observer spinning disk confocal microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Zen software</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Inverted microscope</td><td>Carl Zeiss Inc</td><td>Zeiss Axiovert 200M</td><td></td></tr><tr><td>ICCD camera</td><td>Stanford Photonics</td><td>XR-Mega-10EX S-30</td><td></td></tr><tr><td>Spinning disk confocal scan-head</td><td>Yokogawa Corporation</td><td>CSU-10b</td><td></td></tr><tr><td>Imaris</td><td>Bitplane</td><td></td><td></td></tr><tr><td>mManager</td><td>Vale lab, UCSF</td><td></td><td>Open-source software</td></tr></tbody></table>

ex vivo live imaging of lung metastasis and their microenvironment

转移是癌症相关的发病率和死亡率的一个主要原因。转移是一个多步骤的过程，并且由于其复杂性，支配转移性传播和增长的确切细胞和分子过程仍然难以捉摸。实时成像使细胞微环境的动态性和空间相互作用的可视化。实体瘤通常转移至肺部。然而，肺的解剖位置姿势到活体成像的一个挑战。这个协议提供了用于离体肿瘤细胞和肺转移中其周围基质之间的动态相互作用的实时成像的相对简单和快速的方法。使用这种方法，在其微环境的癌细胞的癌症细胞和基质细胞之间的运动，以及相互作用可以实时可视化数小时。通过使用转基因荧光报道小鼠，荧光细胞系，可注射荧光标记的分子和/或抗体，肺微环境的多个组件可以被可视，如血管和免疫细胞。到图象的不同类型的细胞，一旋转盘共聚焦显微镜，它允许快速，四色图像采集长期连续成像已被使用。从收集到的多个位置和焦平面图像编译时间推移电影显示活转移性和免疫细胞之间的相互作用至少4小时。这种技术可以进一步用于测试化疗或靶向治疗。此外，这种方法可以适用于其它肺相关疾病可能影响肺微环境的研究。

使用纺丝盘共焦显微镜，各种小鼠模型系统和注射剂，转移微环境可以可视化并随时间跟踪。使用MMTV-PyMT; ACTB-ECFP;的c-FMS-EGFP三重转基因小鼠模型，不同的细胞组分的荧光标记（图2A，电影1）。肺实质的典型结构可以在CFP通道由于所有细胞中的β肌动蛋白启动子下表达的ECFP可视化。这些表现为大部分肺组织结构中细胞的大/多肺转移是很容易解决。髓细胞可视化在...

Watch this Scientific Journal Video about Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment at JoVE.com

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

体外肺转移及微环境的实时成像

我们描述用于离体肺转移中的肿瘤细胞-基质相互作用的实时成像的相对简单的方法，利用荧光报告小鼠。使用纺丝盘共焦显微镜，该技术使活细胞的可视化的至少4小时，并可以适用于研究其它炎性肺部疾病。

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

ex-vivo-live-imaging-of-lung-metastasis-and-their-microenvironment

Research

JoVE Journal

Medicine

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

12.3K 次观看.  University of California. 我们描述用于离体肺转移中的肿瘤细胞-基质相互作用的实时成像的相对简单的方法，利用荧光报告小鼠。使用纺丝盘共焦显微镜，该技术使活细胞的可视化的至少4小时，并可以适用于研究其它炎性肺部疾病。 

视频: Ex vivo Live Imaging of Lung Metastasis and Their 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

Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung colonization in osteosarcoma (OS) by fluorescence microscopy. This article provides a detailed description of the protocol, and discusses examples of obtaining image data on metastatic growth using widefield or confocal fluorescence microscopy platforms. The flexibility of the PuMA model permits researchers to study not only the growth of OS cells in the lung microenvironment, but also to assess the effects of anti-metastatic therapeutics over time. Confocal microscopy allows for unprecedented, high-resolution imaging of OS cell interactions with the lung parenchyma. Moreover, when the PuMA model is combined with fluorescent dyes or fluorescent protein genetic reporters, researchers can study the lung microenvironment, cellular and subcellular structures, gene function, and promoter activity in metastatic OS cells. The PuMA model provides a new tool for osteosarcoma researchers to discover new metastasis biology and assess the activity of novel anti-metastatic, targeted therapies.

The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.

应用肺转移法研究骨肉瘤转移性肺定植的实用思考

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

肺定植法在肺部组织循环肿瘤细胞可视化中的建立

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of micrometastases by radiologic imaging and the success in their therapeutic eradication remain limited.
While animal models have proven to be invaluable tools for cancer research1, the monitoring/visualization of micrometastases remains a challenge and inaccurate evaluation of metastatic spread in preclinical studies potentially leads to disappointing results in clinical trials2. Consequently, there is great interest in refining the methods to finally allow reproducible and reliable detection of metastases down to the single cell level in normal tissue. The main focus therefore is on techniques, which allow the detection of tumor cells in vivo, like micro-computer tomography (micro-CT), positron emission tomography (PET), bioluminescence or fluorescence imaging3,4. We are currently optimizing these techniques for in vivo monitoring of primary tumor growth and metastasis in different osteosarcoma models. Some of these techniques can also be used for ex vivo analysis of metastasis beside classical methods like qPCR5, FACS6 or different types of histological staining. As a benchmark, we have established in the present study the stable transfection or transduction of tumor cells with the lacZ gene encoding the bacterial enzyme &beta;-galactosidase that metabolizes the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to an insoluble indigo blue dye7 and allows highly sensitive and selective histochemical blue staining of tumor cells in mouse tissue ex vivo down to the single cell level as shown here. This is a low-cost and not equipment-intensive tool, which allows precise validation of metastasis8 in studies assessing new anticancer therapies9-11. A limiting factor of X-gal staining is the low contrast to e.g. blood-related red staining of well vascularized tissues. In lung tissue this problem can be solved by in-situ lung perfusion, a technique that was recently established by Borsig et al.12 who perfused the lungs of mice under anesthesia to clear them from blood and to fix and embed them in-situ under inflation through the trachea. This method prevents also the collapse of the lung and thereby maintains the morphology of functional lung alveoli, which improves the quality of the tissue for histological analysis. In the present study, we describe a new protocol, which takes advantage of a combination of X-gal staining of lacZ-expressing tumor cells and in-situ perfusion and fixation of lung tissue. This refined protocol allows high-sensitivity detection of single metastatic cells in the lung and enabled us in a recent study to detect "dormant" lung micrometastases in a mouse model13, which was originally described to be non-metastatic14.

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

The novel protocol reported in the present study allows selective detection of lung metastases at single cell resolution in mice by combined in-situ lung perfusion and fixation and X-Gal staining of lacZ-tagged tumor cells.

肺转移瘤在小鼠体内的组合在单细胞分辨率的可视化改进<em&gt;在原位</em灌注肺组织和X-gal染色<em&gt; lacZ的</em&gt;标记的肿瘤细胞

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of ...

医学

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 ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

结合使用尾静脉转移分析与实时 Vivo 成像，以量化乳腺癌转移殖民化和肺负担

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

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 ...

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple opportunities for therapeutic intervention, and the ability to accurately model them in mice is critical to evaluate their effects. Here, a step-by-step protocol is presented for the establishment of orthotopic primary breast tumors and the subsequent monitoring of the establishment and growth of metastatic lesions in the lung using in vivo bioluminescence imaging. This methodology allows for the evaluation of treatment or its biological effects along the entire range of metastatic development, from primary tumor escape to outgrowth in the lungs. Breast orthotopic tumors are generated in mice via injection of a luciferase-labeled cell suspension in the 4th mammary gland. Tumors are allowed to grow and disseminate for a specific amount of time and are then surgically resected. Upon resection, spontaneous lung metastasis is detected, and the growth over time is monitored using in vivo bioluminescence imaging. At the desired experimental endpoint, lung tissue can be collected for downstream analysis. The treatment of established, clinically evident metastasis is critical to improve outcomes for stage IV cancer patients, and it can be evaluated through tail vein models of experimental lung metastasis. However, metastatic dissemination occurs early in breast cancer, and many patients have latent, subclinical disseminated disease after surgery. Utilization of spontaneous models such as this one provides the opportunity to study the whole spectrum of the disease, especially the systemic effects driven by treatment of the primary tumor such as pre-metastatic niche priming, and evaluate treatments on dormant and subclinical disease after surgery.

<meta charset="utf8"></head><body>体内成像测量原位注射乳腺肿瘤细胞的自发性肺转移

The manuscript describes a methodology for the establishment as well as longitudinal growth monitoring of spontaneous lung metastasis from orthotopically-injected breast tumors, amenable to intervention at all stages of the metastatic cascade.

体外肺转移及微环境的实时成像

摘要

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此视频中的章节

长期高分辨率活体显微镜在肺与真空稳定成像窗口

应用肺转移法研究骨肉瘤转移性肺定植的实用思考

肺定植法在肺部组织循环肿瘤细胞可视化中的建立

肺转移瘤在小鼠体内的组合在单细胞分辨率的可视化改进

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

结合使用尾静脉转移分析与实时 Vivo 成像，以量化乳腺癌转移殖民化和肺负担

研究癌症转移至肺部的永久窗口

一种用于乳腺肿瘤微环境活体内成像的无标记分割方法

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

体外肺转移及微环境的实时成像

摘要

探索更多视频

此视频中的章节

长期高分辨率活体显微镜在肺与真空稳定成像窗口

应用肺转移法研究骨肉瘤转移性肺定植的实用思考

肺定植法在肺部组织循环肿瘤细胞可视化中的建立

肺转移瘤在小鼠体内的组合在单细胞分辨率的可视化改进

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

结合使用尾静脉转移分析与实时 Vivo 成像，以量化乳腺癌转移殖民化和肺负担

研究癌症转移至肺部的永久窗口

一种用于乳腺肿瘤微环境活体内成像的无标记分割方法

体内研究 影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

体内研究 影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移