Name: in vitro mesothelial clearance assay that models the early steps of ovarian cancer metastasis
Uploaded: 2012-02-17T13:00:00
Description: Watch this Scientific Journal Video about In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis at JoVE.com

我们要感谢哈佛医学院的尼康影像中心，尤其是珍妮弗水域，拉拉Petrak和温迪三文鱼，培训和使用他们的RSS 2.0订阅显微镜。我们也想感谢罗莎·吴和阿希姆·贝瑟有价值的讨论。这项工作是由NIH资助5695837（M. Iwanicki）GM064346来JSB的支持;由授予博士仪和Sheldon G.阿德尔森医学研究基金会（JSB的）。

1。卵巢癌的细胞球体的形成<ol><li> RFP的表达卵巢癌的细胞培养在10％的基础培养基（自定义的细胞培养液中含有199和一个MCDB105 50:50的混合物，10％的灭活胎牛血清和1％笔链球菌）。来表达RFP的未标记卵巢癌的细胞，转染质粒含有利福平的细胞和细胞表达RFP的选择。另外，病毒载体可用于瞬时表达荧光蛋白或细胞可以预孵育与红色荧光的细胞追踪染料（Invitrogen公司）。 </li><li>对卵巢癌的球体形?...

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“间皮间隙检测”这里使用的时间推移显微镜监测卵巢癌多细胞球体和间皮细胞单层的相互作用，在很大的空间和时间上的细节。此前，几组8-14端点检测表明，卵巢癌的细胞附着和侵入皮细胞单层。此法是独一无二的，它使用荧光标记的细胞来区分间皮细胞的肿瘤细胞，使这两个细胞群的动态，可以监测整个实验。插的过程中可以实时可视化，并随着时间的推移，皮清除率可以定量测定...

<table border="1"><tbody><tr><td> 试剂 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> OVCA433卵巢癌细胞</td><td></td><td></td><td>礼品从Slamon医师谭维义</td></tr><tr><td> ZT间皮细胞</td><td></td><td></td><td>从谭智因斯的礼物</td></tr><tr><td>中等199 </td><td> Gibco公司</td><td> 19950 </td><td></td></tr><tr><td> MCDB105 </td><td>细胞应用公司</td><td> 117-500 </td><td></td></tr><tr><td>胎牛血清的热灭活</td><td> Gibco公司</td><td> 10082 </td><td></td></tr><tr><td>本 - 链球菌</td><td> Gibco公司</td><td> 15070 </td><td></td></tr><tr><td> 96孔板</td><td>康宁COSTAR </td><td> 3799 </td><td></td></tr><tR&gt; <td>羟乙酯（聚甲基丙烯酸羟乙酯） </td><td> Sigma Aldrich公司</td><td> 192066-25G </td><td>为聚甲基丙烯酸羟乙酯的解决方案，为6mg溶于95％乙醇1毫升聚甲基丙烯酸羟乙酯粉</td></tr><tr><td>乙醇</td><td> pharmco-aaper </td><td> 111ACS200 </td><td>稀释至95％，在卫生署2 0 </td></tr><tr><td>细胞培养罩</td><td> nuaire </td><td> NU-425-300 </td><td></td></tr><tr><td>组织文化的孵化器</td><td> Thermo Scientific的</td><td> 3110 </td><td></td></tr><tr><td>孵化器聚黑马板块</td><td> labline仪器</td><td>帝国三305 </td><td></td></tr><tr><td>台式离心机</td><td>贺利氏</td><td> 75003429/01 </td><td></td></tr><tr><td> 6以及玻璃底菜</td><td> MatTek公司。 </td><td> P06G-1.5的20-F </td><td></td></tr><tr><td>纤维连接蛋白</td><td>西格玛</td><td> F1141-1MG </td><td></td></tr><tr><td> PBS </td><td> cellgro </td><td> 21-040-CV </td><td></td></tr><tr><td> RSS 2.0订阅显微镜： </td><td></td><td></td><td></td></tr><tr><td>显微镜</td><td>尼康</td><td></td><td>钛 - E倒立时间推移电动荧光显微镜完美的对焦系统综合</td></tr><tr><td>镜片</td><td>尼康</td><td></td><td> 20X-0.75数值apeture </td></tr><tr><td>卤素灯透射</td><td>尼康</td><td></td><td> 0.52不适用长工作距离聚光镜</td></tr><tr><td>激发和发射滤波器</td><td>色度在尼康住房单通滤波器</td><td></td><td> GFP的前480/40，EM 525/50 RFP-mCherry前575/50 EM 640/50 </td></tr><tr><td>传输和Epifluoresce光路</td><td>萨特</tD&gt; <td></td><td>智能百叶窗</td></tr><tr><td>线性编码机动阶段</td><td>尼康</td><td></td><td></td></tr><tr><td>冷却电荷耦合器件摄像机的</td><td>滨松</td><td> ORCA的-AG </td><td></td></tr><tr><td>显微镜孵化室温度和CO 2控制</td><td>定制</td><td></td><td></td></tr><tr><td>隔振表</td><td>丰田汽车公司</td><td></td><td></td></tr><tr><td> NIS元素软件</td><td>尼康</td><td></td><td>第3版</td></tr></tbody></table>

in vitro mesothelial clearance assay that models the early steps of ovarian cancer metastasis

卵巢癌是第五次在美国癌症相关死亡的首要原因。尽管积极的初步反应治疗，70％至90％的卵巢癌的妇女开发新的转移，复发往往是致命的2。因此，有必要的，它是了解出现二次转移，以发展中间和晚期卵巢癌的更好的治疗。卵巢癌转移发生恶性细胞脱离原发肿瘤部位和传播整个腹腔。传播的细胞可以形成多细胞集群，或球体，将保持独立，或者植入到机关内的腹腔（图1，电影1）。 腹腔内的器官全部一字排开用一个单一的，连续的，一层4-6间皮细胞（图2）。然而，间皮细胞是从下面缺席腹膜肿块，切除人体肿瘤组织切片3,5-7（图2）的电子显微镜研究发现。这表明，由一个未知的过程中，间皮细胞排除从下面的肿块。 以前在体外实验表明，原发性卵巢癌的细胞更有效地附加比8间皮细胞外基质，最近的研究表明，原发性腹膜间皮细胞，实际上提供了一个屏障，卵巢癌的细胞粘附和侵袭（相比，黏附和侵袭不包括与间皮细胞）9,10基板上。这表明，间皮细胞对卵巢癌转移的障碍行动。卵巢癌细胞突破这一障碍，排除间皮有细胞和分子机制，直到最近，仍然不明。 在这里，我们描述日e为在体外试验方法，模型与卵巢癌的细胞球体和间皮细胞在体内的相互作用（图3，电影2）。我们的协议是改编自先前所描述的方法，分析卵巢肿瘤细胞的相互作用与8-16皮单层，并首次在报告中显示，卵巢肿瘤细胞利用整合依赖激活的肌球蛋白和牵引力，以促进排除描述从下间皮细胞肿瘤球体17。这种模式需要时间推移荧光显微镜的优势，在实时监控的两个细胞群，提供了空间和时间上的互动信息。卵巢癌的细胞表达红色荧光蛋白（RFP），而间皮细胞表达绿色荧光蛋白（GFP）。 RFP的表达卵巢癌的细胞球体连接到表达GFP的皮单层。球体的蔓延，侵入，迫使除了创造一个洞单层间皮细胞。这个洞是在GFP图像为负空间（黑）的可视化。洞的面积，然后可以测量，控制和卵巢癌和/或间皮细胞的实验种群之间的间隙活动的差异定量分析。此法只需要一个小数目，卵巢癌的细胞（100细胞每球体X 20-30％条件下球体），所以它是可行的，执行此法，采用珍贵的原发肿瘤细胞样本。此外，该法可以很容易地适应高通量筛选。

Watch this Scientific Journal Video about In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis at JoVE.com

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

这里描述的皮间隙检测，利用荧光标记的细胞和延时视频显微镜，可视化和定量测量卵巢癌多细胞球体和间皮细胞单层的相互作用。此法模拟卵巢癌转移的早期步骤。

这些模型在体外间皮间隙检测卵巢癌转移的早期步骤

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

Research

JoVE Journal

Medicine

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Experimental Metastasis Assay

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

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

MAME的模型四维活细胞成像肿瘤微环境的相互作用，影响的恶性进展

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

利用磁共振成像，容积电脑断层扫描及超音波的裸鼠乳腺癌骨转移大鼠模型血管生成的多模态成像

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

异型三维<em在体外</em&gt;卵巢癌的发生和发展过程中的间质 - 上皮细胞相互作用模型

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

Models of Bone Metastasis

骨转移模型

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

在实时的间皮细胞卵巢癌球体附件和入侵评估

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

在卵巢癌肿瘤起始细胞体外富集

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Three-Dimensional 3D Tumor Spheroid Invasion Assay

三维（3D）肿瘤球体侵袭试验

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

在体内和体外途径研究银河系结构现货卵巢癌转移定植腹膜脂肪

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

腹膜内卵巢癌转移的定量

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...