由美国国家癌症研究所资助1R03CA172923的部分支持。

伦理声明:在研究中使用的小鼠只有在它可能会推动基础生物学或对疾病的治疗最终人类认识的实例可以接受的。 1.订购<ol><li>购买的雌性C57BL / 6小鼠（6岁 - 8周），每组5只小鼠。允许几天的老鼠进行调整。 </li></ol> 2. B16-BL6细胞的制备<ol><li>在无菌罩中删除从37度培养箱和地点细胞培养板。板材应该是大约70汇合与B16-BL6小鼠黑色素瘤细胞％。更大的融合可能会改变细胞的转移潜能。 </li><li>加入1毫升0.05％的胰蛋白酶-EDTA每板，让坐在1分钟，然后吸出胰蛋白酶的培养基溶液。 </li><li>加入1ml每板胰蛋白酶，并返回板的孵化10分钟。 </li><li>从孵化器中移动板到无菌罩后，加入4毫升的无血清罗斯维尔公园纪念研究所Mediu的米（RPMI），以每块板。 </li><li>收集用无菌，机动吸管细胞。喷射细胞对所述板的底部，以在非常微小的角度按下尖，打散细胞。为了确保细胞团块，否则可能堵塞的喷射针的足够的分离重复数次。回忆并转移到50毫升锥形管中。 </li><li>使用血细胞计数器确定细胞密度。保持B16-BL6细胞的总数不变，确定无血清RPMI的体积需要达到0，0.065，0.015，0.25和0.5百万个细胞/ ml的最终浓度。 </li><li>同样等分在50ml管中的介质到15ml锥形管中。离心锥形管2250 XG 5分钟。倒出介质。添加无血清的RPMI适当体积，并确认通过血细胞计数器所需的细胞密度。通过添加额外的RPMI或纺丝和在一个较小的体积 - 相应地重新悬浮调整densities-。 </li><li>分装500微升的各细胞悬浮液到在冰上标记1.8毫升管中。 </li></ol> 3.尾静脉注射<ol><li>拿鼠标，尾巴举行，引导它后，首先进入鼠标限制器。与在阻挡器的主腔室中的躯干，与外面的尾部，拉鼠标朝向限制器的端部。将阻挡柱塞，继续推动，直到鼠标是安全的。 </li><li>旋转鼠标90度，使得侧面尾静脉中的一个被朝上。使用醇垫，大力清洁鼠标的尾部的一侧，特别是在尾静脉是最明显的。 注:良好的照明是对B57BL / 6J小鼠有效尾静脉可视化的关键。热灯或加热手术垫，而不是必须的，也将通过增加尾静脉的体积帮助。 </li><li>在无菌培养罩，从200万个细胞/毫升管收集300μl的细胞培养基。反转针，弹在SIDE和推动柱塞，以从注射器排出气泡。喷射培养的RPMI以导致250微升的最终体积。 </li><li>延长小鼠尾巴与非惯用手。持用非优势手的食指升高的尾部的近端，和用拇指按下尾部的远侧部分的尾部。 注意:在这种方式中，尾静脉在侧向位置上，平行于该阻挡表面上，在尾部的更远侧部分的全等条目可能。 </li><li>针插入尾静脉，朝向远端，在一分钟向下的角度，首先，然后调整针以匹配在进一步插入尾静脉的角度（降低相对注射器的柱塞端的针） 。 </li><li>插入针大约1cm到尾静脉。插入之后，开始推动柱塞，持针稳定。 注意:如果该针在尾静脉注射针的端部是正OT阻挠媒体应不受阻碍地流入静脉。有效喷射的特征在于从静脉血液清除。如果有阻力，或气泡开始形成在注射部位，取出针头，并在沿尾静脉更近侧位置再次尝试。 </li><li>从静脉取出针并丢弃。持反对纱布入口点停止任何出血。 </li><li>从阻挡器取出柱塞和将鼠标在收件人笼。 </li><li>重复所有其它浓度。 注意:肺癌病灶建立大约需要2 - 3周，与取决于细胞系和灶的期望尺寸可变性9在过渡期间，小鼠应为可能保证早期安乐死，如过度喘气症状来监测。 </li></ol> 4.计数肺灶<ol><li>通过在CO 2室设置并暴露于100％的CO 2安乐死鼠标</sUB&gt;以每分钟室体积10-30％引入5分钟。用颈椎脱位跟随。 </li><li>上张开泡沫塑料鼠标。用手术剪，创建沿胸骨手术切口。 </li><li>使两个附加切口，无论从胸骨切口的顶部和分支朝向鼠标的肩膀上，露出了肋骨。 </li><li>使两个切口，每个沿着胸骨的侧面，以从躯干移除左肋，露出了肺和心脏。 </li><li>拿着手术镊子的心脏，切开食道和气管，从鼠​​标释放心脏和肺部。将解剖显微镜下，在放大10倍，以便更好地观察。 </li><li>从肺解剖的心脏，取出胸腺，只留下两个肺。 </li><li>随着解剖显微镜下肺，开始计数肺部病灶。每个病灶应显示为肺部的表面上的圆形，棕色obtrusion。 <ol><li>对于小鼠之间的一致性，计数一个波瓣的表面上灶的数目，然后反转该叶。做四个波瓣单独允许更容易计算。 </li></ol></li><li>保存并执行，如果IHC修复肺，否则丢弃的遗体。 </li></ol>

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</ol>

作者宣称，他们没有竞争的经济利益。

从尾静脉注射循环肿瘤细胞代表了逃避的主要生长部位的转移，并通过像血液或淋巴系统线路迁移，偶尔在远的地方的毛细血管床立业。14上述协议，充当次要的典范转移。注射B16-BL6细胞的最佳数量，实验者可以确定施用药物或免疫细胞群，中和后的相对影响，对癌细胞转移。 该协议有其局限性。的循环肿瘤细胞已被设定为它们转移的能力，因此非免疫原性，具有主要组织相容性复合物1类分子的水平较低。12此外，突发性和聚集大量引进转移不同于患者典型方案其中，转移性肿瘤细胞的数量少消散在从原发肿瘤位置的长时间。此外，静脉注射引进肿瘤细胞组织培养而不是原发肿瘤的起源。因此，这些细胞不像在细胞粘附，迁移，免疫识别和接受器官建立改变导致二次转移。15,16 另一种协议将是B16 / BL6的皮下注射原发性肿瘤的产生和由此产生的"自发"肺转移分析。这些"自发的"转移已发现含有比它们的"实验"对应物更异质性，更加紧密地匹配的那些人类患者。该协议被限制在其确定在转移的实验化合物的影响能力。有横向尾静脉注射，注射到血流中的黑色素瘤细胞的数目可以通过ħ近似emocytometer。与"自发"转移，但是，存在的肿瘤之间的更大的异质性，增加一个额外的变量，从而导致肺病灶的数目。16 总之，尾静脉B16-BL6小鼠恶性黑色素瘤模型表示用于确定转移治疗或免疫治疗的影响的简单模型。通过静脉注射循环肿瘤细胞，研究人员可以复制，在一定程度上，病人转移灶后。考虑转移性肿瘤细胞在癌症患者的破坏性影响，这种模式是理解转移的多步过程的有力工具。

转移是导致死亡的患者恶性肿瘤中的一个主要原因。癌细胞转移传播过程知之甚少，但似乎涉及几个步骤，包括在转移，适应的部位的循环系统内的相邻组织的侵袭，血管内进入淋巴管和血管，存活和转移，外渗从血管新网站的新的微环境，和殖民化在通过增殖和形成继发肿瘤的新网站。1每个生物事件涉及转移性肿瘤细胞的转化，传播和生存。例如，肿瘤细胞的上皮表型转变成间充质表型，加上与胞外基质的成功相互作用，使它们能够侵入邻近组织并转移到身体的其他部位。2,3此外，这些转移细胞最后，循环血液内必须生存和回避来自宿主的免疫监视。4,5，当在体内的远端部位植入，肿瘤细胞必须适应其微环境，以增殖和形成继发性肿瘤。6,7-因此虽然转移是癌症患者中常见的现象，转移性肿瘤细胞具有适合进行治疗干预的脆弱的多个区域。 给予黑素瘤的免疫原性性质，以及最近在免疫疗法的兴趣，型号为黑素瘤越来越有用。8仅在美国，黑素瘤是每年估计9000例死亡的原因。9黑素瘤转移的普通位置是骨骼，大脑，肝和肺。大部分转移到遥远的站点通过血液发生。在血液中循环的肿瘤细胞必须逃避免疫清除，到达远端器官的毛细血管床和通过血管的内皮细胞，以便成功地建立自己的入侵。4-7,10，11 为了模仿转移的常见和致命的现象，鼠B16-BL6细胞系建立。亲本的C57BL / 6黑色素瘤细胞株B16-F0的死者，B16-BL6是用于从静脉注射（导致B16-F10）肺转移10个连续的选择，接着为膀胱膜渗透6连续选择的最终产物。 12因此，它是用于在小鼠建立转移灶的一可靠的黑色素瘤细胞系，特别是当静脉内注射13 足够量的B16-BL6细胞的成B57BL / 6小鼠的尾静脉中，接着的两个或更多周，以允许注入和B16 / BL6细胞的生长注射后，转移灶将形成在肺部。安乐死和检查通过解剖显微镜，数单个病灶存在可以量化的。这，反过来，可用于建立剂量 - 转移效果，如注射B16-BL6细胞的数目与形成在肺部的表面上灶的数目相关。这种模式被称为其中将已知转移性细胞被直接引入到血液中，有利于在肺，肝，或其他调查机关一个快速和可预测的传播和建立"实验性转移"。这是相对于"自发转移"，其中的肿瘤细胞被植入，经常皮下，和转移发起从有机棚癌细胞。14,15 它注入B16 / BL6细胞的适当量入小鼠的尾静脉是重要的。太多了，和肺部将转移支付，而邻近的焦点将是无法区分彼此。太少，和治疗剂的影响将是无形由于固有VARI注射之间ations。为了获得在C57BL / 6小鼠的肺病灶的最佳数目，有必要在考虑到个体的区分性既定转移灶的肺中的表面上的量关联注射细胞的数目灶。该协议将展示C57BL / 6小鼠转移性小鼠黑色素瘤模型。

<table><tbody><tr><td>Trypsin</td><td>Corning</td><td>MT25052CV</td><td></td></tr><tr><td>RPMI 1640 Medium</td><td>Corning</td><td>MT15040CM</td><td></td></tr><tr><td>Phase Contraast Hemacytometer</td><td>Hausser</td><td>02-671-54</td><td></td></tr><tr><td>Micro-Fine IV Insulin Syringes</td><td>BD</td><td>14-829-1D</td><td></td></tr><tr><td>Sterile Alcohol Prep Pads</td><td>Fisherbrand</td><td>22-363-750</td><td></td></tr><tr><td>Mouse Restrainer</td><td>Braintree Scientific</td><td>NC9999969</td><td>Restrainer choice depends on age/size of mice</td></tr><tr><td>Heating Pad</td><td>Harry Schein</td><td>NC0012697</td><td>Optional</td></tr></tbody></table>

injection of syngeneic murine melanoma cells to determine their metastatic potential in the lungs

Approximately 90% of human cancer deaths are linked to metastasis. Despite the prevalence and relative harm of metastasis, therapeutics for treatment or prevention are lacking. We report a method for the establishment of pulmonary metastases in mice, useful for the study of this phenomenon. Tail vein injection of B57BL/6J mice with B16-BL6 is among the most used models for melanoma metastases. Some of the circulating tumor cells establish themselves in the lungs of the mouse, creating "experimental" metastatic foci. With this model it is possible to measure the relative effects of therapeutic agents on the development of cancer metastasis. The difference in enumerated lung foci between treated and untreated mice indicates the efficacy of metastases neutralization. However, prior to the investigation of a therapeutic agent, it is necessary to determine an optimal number of injected B16-BL6 cells for the quantitative analysis of metastatic foci. Injection of too many cells may result in an overabundance of metastatic foci, impairing proper quantification and overwhelming the effects of anti-cancer therapies, while injection of too few cells will hinder the comparison between treated and controls.

经目测，在灶面的变化是显而易见的。在解剖显微镜下计数肿瘤的数目应该得到注射肿瘤细胞的数量和所得的数目和可数焦点之间的线性关系。使用注射细胞的最佳数量，目标是其中肺部不完全覆盖，因为它们是在图1A中500,000个细胞/毫升，并没有太稀疏覆盖，因为它们是在62,500个细胞/ ml和125000细胞/毫升。这也是由线性相关（ 图1B）量化。 <img alt="图1" src="/files/ftp_upload/54039/54039fig1.jpg" /> 图1.龙灶尾静脉注射所得B16 / BL6细胞。 （A）的肺病灶可以肺从C57BL / 6小鼠作为褐色圆块（下箭头代表灶）的表面上被可视化。由于该窝点注入的细胞培养（每幅图像以上）的性增加，所以不造成肺部病灶（每张图片下方）的相应的数字。从小鼠注射细胞密度最高的肺部有病灶肺的最大可视范围。刻度条表示为20毫米。（B）中相对于所述细胞培养物密度的肺病灶的计数。上面有125000个/ ml近似线性关系，由血球确定。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54039/54039fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs at JoVE.com

Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

Metastasis plays a profound role in the virulence of cancer, accounting for an estimated 90% of deaths. We report a protocol for a metastatic melanoma model in mice that is useful for determining the efficacy of therapeutic agents against this clinical phenomenon.

同基因小鼠黑色素瘤细胞注射，以确定其在肺转移潜能

Approximately 90% of human cancer deaths are linked to metastasis.  Despite the prevalence and relative harm of metastasis, therapeutics for treatment or ...

injection-syngeneic-murine-melanoma-cells-to-determine-their

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

17.6K 次观看.  Beth Israel Deaconess Medical Center & Harvard Medical School. Metastasis plays a profound role in the virulence of cancer, accounting for an estimated 90% of deaths. We report a protocol for a metastatic melanoma model in mice that is useful for determining the efficacy of therapeutic agents against this clinical phenomenon.

视频: Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

实验转移的含量

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

科研

医学

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 ...

癌症研究

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

肿瘤细胞外侧尾静脉注射后肺转移的病理分析

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

Analysis of Lymph Node Volume by Ultra-High-Frequency Ultrasound Imaging in the Braf/Pten Genetically Engineered Mouse Model of Melanoma

Tyr::CreER+,BrafCA/+,Ptenlox/lox&#160;genetically engineered mice (Braf/Pten mice) are widely used as an in vivo model of metastatic melanoma. Once a primary tumor has been induced by tamoxifen treatment, an increase in metastatic burden is observed within 4-6 weeks after induction. This paper shows how Ultra-High-Frequency UltraSound (UHFUS) imaging can be exploited to monitor the increase in metastatic involvement of the inguinal lymph nodes by measuring the increase in their volume.
The UHFUS system is used to scan anesthetized mice with a UHFUS linear probe (22-55 MHz, axial resolution 40 &#181;m). B-mode images from the inguinal lymph nodes (both left and right sides) are acquired in a short-axis view, positioning the animals in dorsal recumbency. Ultrasound records are acquired using a 44 &#181;m step size on a motorized mechanical arm. Afterward, two-dimensional (2D) B-mode acquisitions are imported into the software platform for ultrasound image post-processing, and inguinal lymph nodes are identified and segmented semi-automatically in the acquired cross-sectional 2D images. Finally, a total reconstruction of the three-dimensional (3D) volume is automatically obtained along with the rendering of the lymph node volume, which is also expressed as an absolute measurement.
This non-invasive in vivo technique is very well tolerated and allows the scheduling of multiple imaging sessions on the same experimental animal over 2 weeks. It is, therefore, ideal to assess the impact of pharmacological treatment on metastatic disease.

Melanoma is a very aggressive disease that quickly spreads to other organs. This protocol describes the application of ultra-high-frequency ultrasound imaging, coupled with 3D rendering, to monitor the volume of the inguinal lymph nodes in the Braf/Pten mouse model of metastatic melanoma.

超高频超声成像分析黑色素瘤Braf/Pten基因工程小鼠模型中的淋巴结体积

Tyr::CreER+,BrafCA/+,Ptenlox/lox&#160;genetically engineered mice (Braf/Pten mice) are widely used as an in vivo model of metastatic melanoma. Once a ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

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

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

人类前列腺癌转移的原位小鼠模型

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

A Robust Discovery Platform for the Identification of Novel Mediators of Melanoma Metastasis

Metastasis is a complex process, requiring cells to overcome barriers that are only incompletely modeled by in vitro assays. A systematic workflow was established using robust, reproducible in vivo models and standardized methods to identify novel players in melanoma metastasis. This approach allows for data inference at specific experimental stages to precisely characterize a gene's role in metastasis. Models are established by introducing genetically modified melanoma cells via intracardiac, intradermal, or subcutaneous injections into mice, followed by monitoring with serial in vivo imaging. Once preestablished endpoints are reached, primary tumors and/or metastases-bearing organs are harvested and processed for various analyses. Tumor cells can be sorted and subjected to any of several 'omics' platforms, including single-cell RNA sequencing. Organs undergo imaging and immunohistopathological analyses to quantify the overall burden of metastases and map their specific anatomic location. This optimized pipeline, including standardized protocols for engraftment, monitoring, tissue harvesting, processing, and analysis, can be adopted for patient-derived, short-term cultures and established human and murine cell lines of various solid cancer types.

This article describes a workflow of techniques employed for testing novel candidate mediators of melanoma metastasis and their mechanism(s) of action.

用于鉴定黑色素瘤转移新介质的强大发现平台

Metastasis is a complex process, requiring cells to overcome barriers that are only incompletely modeled by in vitro assays. A systematic workflow was ...

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

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple ...

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

原发灶和MEF细胞分离，研究肺转移

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

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 ...

同基因小鼠黑色素瘤细胞注射，以确定其在肺转移潜能

摘要

探索更多视频

实验转移的含量

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

肿瘤细胞外侧尾静脉注射后肺转移的病理分析

超高频超声成像分析黑色素瘤Braf/Pten基因工程小鼠模型中的淋巴结体积

人类前列腺癌转移的原位小鼠模型

用于鉴定黑色素瘤转移新介质的强大发现平台

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

原发灶和MEF细胞分离，研究肺转移

体外肺转移及微环境的实时成像

同基因小鼠黑色素瘤细胞注射，以确定其在肺转移潜能

摘要

探索更多视频

实验转移的含量

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

肿瘤细胞外侧尾静脉注射后肺转移的病理分析

超高频超声成像分析黑色素瘤Braf/Pten基因工程小鼠模型中的淋巴结体积

人类前列腺癌转移的原位小鼠模型

用于鉴定黑色素瘤转移新介质的强大发现平台

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

原发灶和MEF细胞分离，研究肺转移

体外肺转移及微环境的实时成像

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