由美国国立卫生研究院资助P30 CA051008和T32 CA009686（ATR）的支持。

1.球体产生的<ol><li>制备单细胞悬浮液通过分离用的PBS 70％汇合的贴壁〜癌细胞培养物悬滴培养洗涤随后暴露于0.05％的胰蛋白酶-EDTA溶液。 <ol><li>中和与细胞培养基的胰蛋白酶溶液和计数用细胞悬浮液的等分试样的细胞。注意:特定细胞培养基将取决于细胞系进行测试。遵循ATCC建议媒体，其中该细胞培养基是通常的DMEM + 10％FBS。更多信息可在材料表中找到。 </li><li>执行稀释，以允许每细胞培养基的20微升一滴500-1,000细胞的播种。 </li></ol></li><li>收购10cm皿加入5毫升无菌PBS的底部。注:此步骤可以防止蒸发悬滴。成本较低的"细菌学级"菜可以代替细胞培养级的菜肴中使用，因为细胞不会续作用在培养表面。 </li><li>使用多通道移液管到稀释的细胞悬浮液20微升的液滴转移到盖子的内表面上。 <ol><li>吸取40滴（5排8滴）到10 cm培养皿的盖子。 </li></ol></li><li>倒置的盖子，将在培养皿。孵育悬滴培养物在37℃下72小时，以产生球体。 <ol><li>翻转盖的自信，但控制的方式。注意:反相盖子过快或过慢会引起液滴移动。某些细胞系可以形成在48小时或更少的球状体，而其他的可能需要超过72小时，以产生紧凑的聚集体。 </li></ol></li></ol> 2.球体嵌入到3D矩阵<ol><li>嵌入球体之前过夜解冻的生长因子减少的基底膜材料的等分试样于4℃。 <ol><li>可选:图层井ECM提前预防与组织文化前进潜力球体的互动再面。 <ol><li>吸取200微升每孔ECM在24孔板。 </li><li>倾斜板，以确保对ECM覆盖整个井表面。 </li><li>小心地除去多余的ECM用吸管。 </li><li>孵育板直到井干:3小时在室温或过夜，在4℃。 </li></ol></li></ol></li><li>通过倾斜的组织培养皿的盖子，汇集媒体收集的球体。与球体的媒体转移到1.5 ml离心管。 </li><li>允许10分钟的球体，收于离心管的底部。球状​​体应该是肉眼可见的。 </li><li>混合100微升的基底膜材料用100μl冷的（4℃）的I型胶原蛋白在一个单独的预冷管，保持该混合物在4℃下 ，以防止任一凝胶从固化过早。 <ol><li>使用预冷枪头，如果转让的小卷。 </li><li>提供医疗服务时，混合基底膜材料和I型胶原，以防止空气气泡的形成。注: 气泡后可能妨碍成像的协议，如果他们成为镶嵌在凝胶。 </li></ol></li><li>吸从离心管的40微升底部的球状体，并结合基底膜材料/ I型胶原混合物。要小心，以防止形成气泡混合时。注:该解决方案包含100微升基底膜材料，100微升I型胶原，和40微升的球体将创造足够的材料为4个独立的3D文化。这可以放大或缩小。 </li><li>吸取40微升滴粘性的混合物的成孔上的24孔平板的中心。保持板水平，以防止该混合物从运行到井的侧面。注:在24孔板中，一列 - 4口井 - 建议对每个要测试的条件。 </li><li>放置板INTOA 37℃培养箱并留下静置30分钟。三维文化将在此期间聚合。 </li><li>慢慢地淹没在3D培养1毫升细胞培养基。 <ol><li>确保将其添加时入井，以进一步促进凝胶的聚合媒体是温暖的。 </li><li>关键的一步: 轻轻将介质添加到 3D文化。吹打媒体太快入井可导致从组织培养表面上的三维培养物的脱离。 </li></ol></li></ol> 3.监测和分析球体入侵<ol><li>图片入侵从球体到周围的3D矩阵，在时间点由研究者决定。获得使用倒置显微镜以20倍的物镜的照片。注意:理想的时间点会取决于细胞系被测试不同。电镀后不久更具侵入性的细胞系将开始从球体的出口，因此，采取初步的照片S内的几个小时后，电镀。一般来说，拍摄照片，在0小时（接种后），24小时和48小时。入侵通常最后24至48小时后，电镀。 </li><li>利用图像分析软件，如图像J.定量侵袭能力<ol><li>分析侵入从旋转椭圆体的边缘的小区距离。 <ol><li>使用顺子工具，以纪念半径和/或最大侵入距离。 </li><li>点击顶部菜单中的"分析"，然后单击"测量"，显示长度测量。 </li></ol></li><li>分析入侵的球体外面总有创面积。 <ol><li>使用徒手绘制工具来跟踪球体和/或总侵入区域的边界。 </li><li>点击顶部菜单中的"分析"，然后单击"测量"，显示面积测量。 </li></ol></li><li>使用类似的投资回报率经理工具插件创建测量堆栈的图像。 </li></ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

本研究评估癌细胞系面板的性能在一个球体侵袭测定（ 表1）。一般地，我们发现，球体的形成增强了更多上皮前瞻性的细胞系，其中细胞 - 细胞连接的存在促进了球体状结构的形成。已知已经历上皮间质转变的细胞系，如MDA-MB-231细胞不形成在最有可能的悬滴培养球状体由于它们的减小E-，N-，和P-cadherin表达16。 一些在球体侵袭测定的步骤需要密切遵守的...

经确定癌症细胞运动是预测转移潜能的，因为这样的行为通过基底膜和进入循环系统1促进侵袭。大多数研究蠕动集中于细胞如何表现在二维（2D）的设置，虽然它变得普遍认识到，细胞在三维（3D）矩阵的运动是更具代表性如何这些相同的细胞将实际的表现在体内 2。三维培养系统正越来越多地用于研究的细胞行为，范围从细胞形态，对生长动力学和药物敏感性3。到3D环境的上下文中监测癌细胞侵袭的愿望已经导致了通过悬滴培养法4，接着通过嵌入这些球体成3D胞涉及三维细胞聚集体（球状体）的产生先前建立的技术合成矩阵（ECM）的胶原蛋白和基底膜材料5组成。该方法旨在通过提供可以很容易地利用在各种实验条件下，比较入侵一个简化的方法来改善这些先前建立的技术。 更传统的方式来评估在体外细胞运动是划痕缠绕法和Transwell小实验6。前者测定描绘细胞运动在二维设置，因此独立的多种功能用于体内侵入关键的， 如蛋白酶活性7。 Transwell小测定可以更好的模型细胞入侵时井刀片涂覆有细胞外基质底物，但是，只有一个参数， 即细胞在相对 ​​膜表面的外观被测量，和细胞侵袭的许多细微差别，因此不是很容易观察到。相对于这些技术中，癌细胞球体侵袭测定（图1 &lt;/强&gt;）允许细胞侵入中的设置，不仅生理学相关的实时监控，而且还允许重要细胞系特有的功能而被可视化，如个人与集体细胞迁移8。这种方法也得到了标准的3D文化生长测定的优点。通过悬滴法细胞聚集的产生最初约束细胞运动，从而使细胞将被诱因侵入后该约束被提升。此外，一旦该约束被抬起，细胞出口将进行在一致的方向，然后可以方便地定量。 用于癌症细胞球体测定中最流行的ECM材料是基底膜和I型胶原，其中每个组件具有在影响转移行为重要和独特的作用。基质胶是由Engelbreth-Holm的群小鼠肉瘤细胞产生的蛋白质的分泌的混合物中，并富集basement的膜蛋白诸如层粘连蛋白，巢蛋白和IV型胶原9。出于这个原因基质胶是以下称为"基膜材料。"这些基底膜材料提供了所需的除了该施加的范围内的效果对细胞行为11许多其它蛋白质癌细胞侵袭10期间整合附着力必需配体。相比较而言，I型胶原，通常从肌腱等致密的胶原结构12的酸消化制备，是在作为结缔组织和间质支持组织的身体和器官的一个主要的结构元件更简单的基质材料。它已被证明胶原的物理特性可以调节的一些细胞运动的特征;例如，胶原纤维在肿瘤基质界面的取向允许癌细胞沿着这些原纤维侵入到基质13时，随后迁移。在这里介绍的试验中，两个I型胶原和基底膜材料被用作工具来研究三维癌细胞 - 基质相互作用。 抑制控制侵入可监控的细胞已经嵌入在三维矩阵后途径的或刺激的效果。细胞可以生长在挂滴剂或经转移到三维培养，这取决于一个漫长的处理是否需要调节入侵期间进行预处理。对于更短的处理，建议该药物可以与采集后的球体悬浮液，以及将包围3D培养，以促进适当的药物暴露于细胞的培养基混合。接着，正常或肿瘤相关的基质细胞可以混合与基质材料，以评估其在调节肿瘤细胞侵袭的作用，或以确定如何旁分泌和自分泌信号影响细胞的行为。这个想法是在一项研究显示，其中山坳共培养对癌细胞和内皮细胞在悬滴导致球状体14内的血管网。最后，将ECM成分也可以改变，如癌细胞的侵袭是通过不同的基板15的影响。下面提出的方法将由此提供用于评估在各种条件癌细胞侵袭的框架。一般来说我们发现，并非所有的细胞系将在悬滴创建球状体和上皮前瞻性细胞系通常形成定期球体。

<table>
	<tbody>
		<tr>
			<td>Matrigel Growth Factor Reduced</td>
			<td>Corning</td>
			<td>CB-40234</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen Type I, Rat Tail, 100 mg</td>
			<td>Millipore</td>
			<td>08-115</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>11995-065</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium&#160;</td>
			<td>Life Technologies</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Life Technologies</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, Heat Inactivated</td>
			<td>Omega Scientific</td>
			<td>FB-12</td>
			<td></td>
		</tr>
		<tr>
			<td>100mm TC-treated Dishes</td>
			<td>Corning Incorporated</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well TC-treated Plates</td>
			<td>NEST Biotechnlology</td>
			<td>702001</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus IX-71 Inverted Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Cell lines were maintained in DMEM + 10% FBS, with the expection of BT-474 and LNCaP cells, which were mantained in RPMI + 10% FBS.</td>
		</tr>
	</tbody>
</table>

a cancer cell spheroid assay to assess invasion in a 3d setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

使用球体侵袭测定（图1），肿瘤细胞系的组为他们以形成球状体，以及用于小区出口的在3D基质植入后表现出组成的基底膜材料和I型胶原蛋白的量的能力进行了测试（ 表1）。这些结果表明，并不是每一个细胞系将产生良好的球状体，其中细胞系具有体外上皮形态倾向于产生定期和光滑聚集体。不同的细胞系在测定侵入的能力也是可变的。已经建立的作为是侵入性...

Watch this Scientific Journal Video about A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting at JoVE.com

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

癌细胞球体检测，以评估入侵的3D设置

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...

a-cancer-cell-spheroid-assay-to-assess-invasion-in-a-3d-setting

Research

JoVE Journal

Medicine

32.4K 次观看.  Georgetown University. This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored. 

视频: A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.
Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.
The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.

This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.

一个实时的基于电阻抗技术测量癌细胞入侵内皮细胞单层

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of ...

科研

医学

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

球体的检测，以测量TGF -β诱导的入侵

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

3维建模和成像集体细胞的侵袭

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

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

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 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

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

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

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 ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

乳腺癌细胞侵袭定量使用三维（3D）模型

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

一种改进<em&gt;在体外</em&gt;入侵检测，以确定激素，在调节肿瘤细胞侵袭的细胞因子和/或生长因子的潜在作用

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

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 ...

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

The Focus Formation Assay provides a straightforward method to assess the transforming potential of a candidate oncogene.

注重形成:一个基于Cell的检测，以确定一个基因的致癌性

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

颈动脉输液的紫杉烷类化合物在小鼠体内的药代动力学和药效学分析

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...