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>对卵巢癌的球体形成之前，有必要准备低96附着力以及圆底培养皿。要产生低附着力文化的板，聚HEMA30μL溶液（1毫升95％乙醇为6mg羟乙酯）被添加到每个井的96康宁细胞培养皿。在37°C非湿润的孵化器，蒸发乙醇，L孵育96孔板eaving对每口井的聚甲基丙烯酸羟乙酯的电影。这种聚甲基丙烯酸羟乙酯的电影阻止细胞附着到井底，迫使18悬浮液中的细胞生长。 [另外，超低的附件培养皿（康宁）可以用聚甲基丙烯酸羟乙酯涂层菜，来代替。] </li><li>低粘附培养皿准备后，一盘卵巢癌的细胞，颗粒细胞，在3分钟，在900离心力台式离心机（贺利氏）吸上清，重新暂停在10％，相应的中等trypsinize。 </li><li>使用血球细胞计数。 </li><li>调整细胞等有50μl的100个细胞的10％，相应的中等浓度。 </li><li>以及每96聚甲基丙烯酸羟乙酯涂层培养皿均匀悬浮稀释细胞悬液50μL。 </li><li>在37℃16小时细胞培养孵化器（这段时间内应增加或减少所花费的时间取决于孵育96孔板一个特定的细胞株，形成多细胞球体所需的实验条件下），使卵巢癌细胞聚集在一起，形成一个单一的每口井的多细胞球体。在此期间可以接受一些肿瘤细胞凋亡，所以重要的是要选择一个时间，诱导细胞凋亡前。 </li></ol> 2。间皮细胞单层形成<ol><li>在细胞培养罩，预涂层井6以及玻璃底部MatTek菜与纤维连接蛋白，加入2mL的每个菜以及一个5μg纤维连接蛋白/毫升PBS溶液，在室温下孵育30分钟。允许高分辨率的显微成像的光学质量的玻璃裤MatTek菜。 </li><li>间皮细胞表达GFP的10％的基础培养基中培养。板皮细胞Trypsinize，旋转，在台式离心机（Heraeus公司）在900RPM 3分钟，吸出上清液，并在10％，相应的中等重新挂起。 “这里所用的皮细胞表达GFP时，他们得到的，但可以通过质粒含有绿色荧光蛋白基因转染，或在绿色荧光的细胞追踪染料（Invitrogen公司）preinc​​ubating细胞产生的未标记的间皮细胞。 </li><li>经过30分钟的纤维连接蛋白孵化（步骤2.1），与2毫升PBS洗的水井的MatTek培养皿。 </li><li>吸干PBS和板6×10 5％以及在每个6以及MatTek菜的井间皮细胞。在37℃的细胞培养孵化器一夜之间让皮细胞菜的重视，并形成了单层孵育MatTek菜。 </li></ol> 3。间皮细胞间隙分析<ol><li>用吸管收集96以及聚甲基丙烯酸羟乙酯涂层板卵巢癌的球体。 </li><li>从6井MatTek菜含有间皮细胞单层吸介质。用2毫升PBS洗一次。新增的96个以及带够所有的球体迟到的MatTek菜（〜3倍的球体，球体降落成像帐户上的不能成像菜，部分）。 </li><li>放置在一个倒置的宽视场荧光显微镜能够执行持续时间至少8小时的时间推移成像阶段MatTek菜。使用机动的舞台形象在菜的多个位置，多个球体插事件，在一个单一的实验。我们用完美的对焦系统集成和低[20X-0.75数值孔径（NA）的]放大倍率/无微分干涉对比（DIC）光学尼康钛 - E倒置电动广角荧光显微镜的时间推移，与0.52不适用尼康卤素灯透射长工作距离（LWD）冷凝器，尼康快（&lt;100毫秒切换时间）激发和发射滤波器（GFP的前480/40，EM 525/50，RFP-mCherry EX EM 575/50 640/50），萨特快速传播和萤光光路智能关机TERS，尼康线性编码机动阶段，滨松ORCA的银冷却电荷耦合器件（CCD）摄像头，一个定制的显微镜，孵化室温度和CO 2控制，尼康NIS元素AR软件版本，并TMC隔振表。 </li><li>卵巢癌的细胞球体将落户盘底部和重视的间皮细胞单层。 GFP，RFP和相位图20 +球体/单层相互作用，每次10分钟，收集8小时。 </li><li> RFP的表达卵巢癌的细胞球体表达GFP创建一个洞单层间皮细胞的单层侵入。 8小时后，测量孔的大小，使用元素的软件（或其他合适的软件，如图像J）在GFP图像跟踪黑洞。孔的大小正常化最初的球体大小，孔的大小，由相应的RFP的图像在时间为零球体的大小除以8小时。在这个前充足的，孔的大小只测一次，但它可以测量整个8小时的实验多次，以便更好地了解动态插。 </li></ol> 4。代表结果在这个例子中，我们比较皮OVCA433卵巢癌的细胞球体，有塔林-1的减毒表达控制OVCA433球体的通关能力。从各组OV​​CA433球体添加到MatTek菜含有征途间皮细胞单层。从每组六个球体成像八个小时，每10分钟（图4 3，电影，电影4）。单层制作传播球体的孔进行测量和平均从每个组6个职位。图4表明，大林1击倒球体创造的平均清关区显着高于控制球体创造的平均面积小，这表明，大林皮间隙由OVCA433卵巢癌的球体。 <img alt="图1" src="/files/ftp_upload/3888/3888fig1.jpg" /> 图1。卵巢癌转移。原发性卵巢肿瘤的发展无论是从卵巢表面上皮或输卵管。肿瘤细胞/集群断绝从原发肿瘤和收集在腹腔。肿瘤细胞可以聚集形成多细胞球体。球体然后附加到腹腔间皮细胞单层衬砌​​。皮细胞被排除从下面所附的卵巢癌的球体，使球体获得底层基底膜。 电影1。卵巢癌转移。 <a href="http://www.jove.com/files/ftp_upload/3888/Movie_1_Lower_Quality.mp4">点击这里观看电影</a> 。 <img alt="图2" src="/files/ftp_upload/3888/3888fig2.jpg" /> 图2。 </strONG&gt;行间皮细胞表面人腹膜组织，并从下方卵巢癌的细胞植入排除。 <img alt="图3" src="/files/ftp_upload/3888/3888fig3.jpg" /> 图3。间皮间隙检测。卵巢癌的球体形成孵化100 RFP的表达卵巢癌的细胞每孔聚甲基丙烯酸羟乙酯涂层96底部以及轮在37°C培养16小时菜。聚甲基丙烯酸羟乙酯防止附着到培养皿中的细胞，使细胞保持悬浮，并坚持到另一个，每口井，形成一个单一集群。镀1纤维连接蛋白涂层以及MatTek菜6X10每口井的5间皮细胞，并在37°C孵育16小时的板皮细胞单层准备。的球体，然后转移到MatTek菜皮单层和两个细胞群成像为8小时，每10分钟使用尼康钛 - E我电动nverted广角荧光显微镜的时间推移和元素的软件。 电影2。间皮间隙检测。 <a href="http://www.jove.com/files/ftp_upload/3888/Movie_2_lower_quality.mp4">点击这里观看电影</a> 。 <img alt="图4" src="/files/ftp_upload/3888/3888fig4.jpg" /> 图4。大林1 OVCA433球体表达的衰减，降低皮的清除能力。大林1减毒表达与不OVCA433球体（红色）被允许附加到并侵入到征途皮单层（绿色）。两个细胞群成像为8小时，每10分钟使用尼康钛 - E倒置电动广角荧光显微镜的时间推移和元素的软件。图表显示，1大林衰减显着降低间皮细胞间隙（位数情节与手段的绿色酒吧）。 电影3。控制OVCA433球体（红色），侵犯到皮单层（绿色）。 <a href="http://www.jove.com/files/ftp_upload/3888/Movie_3.mov">点击这里观看电影</a> 。 电影4。大林1 OVCA433球体表达（红色）的衰减降低皮（绿色），通关能力。 <a href="http://www.jove.com/files/ftp_upload/3888/Movie_4.mov">点击这里观看电影</a> 。

<ol>
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M., You, J. <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+characterization+and+micromechanics+of+tumor+cell+chemotactic+protrusion,+locomotion,+and+extravasation.">In vitro characterization and micromechanics of tumor cell chemotactic protrusion, locomotion, and extravasation.</a> Ann. Biomed. Eng. 30, 344-355 (2002).</li></ol>

我们什么都没有透露。

“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 ...

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Research

JoVE Journal

Medicine

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

14.4K 次观看.  Harvard Medical School. 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。

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

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

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

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

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

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo 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

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

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

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

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

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

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 ...

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  ...

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

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

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

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of 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 ...

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

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 women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

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 ...

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

造型卵巢癌传播在人类腹腔的器官文化的早期步骤

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. ...