The authors acknowledge funding sources from D&#8217;Youville College School of Pharmacy, Buffalo, NY.

1.准备不同的媒体和其他组件<ol><li>事先进行实验，制备介质选自由DMEM或其他指定的介质中，并添加任一正常胎牛血清，木炭剥离的FBS，或木炭剥离的FBS加组件进行测试。需要注意的是多个组件可以在每个实验进行测试。 </li><li>称出并稀释的激素，生长因子，细胞因子或适当地在生理浓度溶解在木炭脱色的血清。 </li></ol> 2.准备胶原基质上的冰<ol><li>制备2毫升胶原I基质中2.2毫克/毫升通过在冰上加入以下无菌过滤组件:加入200μl10倍的PBS（pH为7.4），5.4微升的1N NaOH，600微升双蒸H 2 O为和1.2毫升胶原I （在3.63毫克/毫升）。 </li><li>保持胶原蛋白I溶液在冰上，直到准备板。 </li></ol> 3.准备迁移/侵袭板用于分析&lt;/ P&gt; <ol><li>对于要测试的每个细胞系，使用一种24孔室板，其中12孔含有插入。使用额外的12个孔不含有插入用于将趋化介质和转印刀片为实验的设置。 注:在平板上用聚乙烯对苯二甲酸盐（PET）膜和8微米孔径胶原基质是最适合的细胞系用在这里。然而，在预涂板一个基质胶基质也可以取代的孔径根据细胞系被调查下降。 </li><li>清楚地标注板，采用每条件3口井被分析（FBS迁移，CS-FBS迁移，FBS侵袭，而CS-FBS侵袭以及FBS迁移的控制，非侵入性的细胞系）。通过移动通孔运动中的PET膜，并测定侵袭通过胶原或基质胶基质，然后通过在所述膜孔测定迁移。使用不同的颜色标记为每个小区条件到标识在电镀过程。 </li></ol> 4.分装的入侵矩阵<ol><li>仔细吸取用于入侵检测75微升胶原基质溶液进入插入物。请小心，以避免气泡。通过将一个倒枪头表面驱散气泡。 </li><li>该板用胶原涂覆的刀片转移到37℃和5％CO 2的培养箱中30分钟，使凝胶固化。 </li></ol> 5.板上的细胞上膜或入侵矩阵<ol><li>同时，trypsinize细胞和有10％FBS补充培养基。自旋细胞在200×g离心5分钟，在台式离心机和在无血清培养基冲洗3次。 </li><li>重悬在无血清培养基。计数细胞用血细胞计数器或自动幻灯片计数器。添加无血清培养基以5×10 4个细胞/ ml的终浓度。 </li><li>当胶原基质凝固（30分钟后），加入700μl的媒体无论是2％FBS的定义或活性炭处理的FBS到每个孔中。每盘12个刀片: <ul><li> 3插入有胶原蛋白和井媒体+ 2％FBS </li><li> 3插入有胶原蛋白和井媒体+ 2％CS-FBS </li><li> 3插入没有胶原蛋白和井媒体+ 2％FBS </li><li> 3插入没有胶原蛋白和井媒体+ 2％CS-FBS </li></ul><ol><li>使用取决于被测试的因子的数目和与对应于每个状态的无胶原控制附加平板上。 </li></ol></li><li>添加细胞悬浮液的刀片以5×10 4个细胞/ ml，在所有的迁移和侵袭插入电镀每个刀片500微升的细胞。 </li><li>孵育细胞22小时，在37℃。 </li></ol> 6.量化迁移和侵入细胞的数量<ol><li>使用甲醇固定液，曙红，和苏木，在分开的井孔中的设置染色。 </li><li>用棉签从每孔除去细胞和基质。Rrepeat为每个井第二拭子应用。 </li><li>用钳子，沾每次插入1秒5次到各3解决方案连续。 </li><li>允许插入干O / N。 </li><li>任我）去除过滤器，用手术刀，小心地切割周围的边缘，并安装在盖玻片和浸油的幻灯片，或ii）允许插入干O / N反转，直接使用插入显微镜。 </li><li>第二天，查看幻灯片或插入与20X物镜的显微镜下，并采取5张从过滤器的不同区域。为了提高一致性，需要4外场和一个中心。 </li><li>计数细胞用于使用ImageJ的软件的所有条件，并适用于下面的公式。 </li><li>确定百分入侵如下: 平均＃细胞通过胶原侵入我插入=一 平均＃细胞通过控制插入的迁移= B ％入侵=（A / B）×100 </li><li>确定入侵指数在2％FBS如下: ％侵袭细胞被测定（在2％FBS）= C ％侵袭控制非侵入细胞（2％FBS）= D 浸润指数（FBS）=（C / D） </li><li>确定入侵指数在2％CS-FBS如下: ％侵袭的细胞被检测（在2％CS-FBS）= E ％入侵控制非侵入性的细胞（2％CS-FBS）的= F 入侵指数（CS-FBS）=（E / F） </li></ol> 7.重复实验协议比较活性炭处理FBS为活性炭处理FBS + X n，其中多因素综合<ol><li>如使用不同的组件为"X"或组分的组合需要重复该过程多次。 </li><li>应用计算，以确定各因素"X"的迁移和侵袭影响的贡献。 </li></ol>

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The authors have nothing to disclose.

肿瘤转移是一个多步骤的过程。细胞必须通过基底膜断裂，intravasate成要么淋巴系统或血液微血管，它们被运到远处位点。肿瘤细胞然后外渗和移成macrometastasis 12。通过上皮间质转化（EMT），肿瘤侵袭和转移的进展已增强了类固醇激素13,14，生长因子15-18和细胞因子19-21。这些分子是如此关键，解决肿瘤进展的信号通路，即化验的发展，以解决他们在肿瘤侵入的潜在...

一种新颖的侵袭实验研制了具有确定具体激素，细胞因子和/或生长因子如癌细胞侵袭化学引诱物的参与的目标。肿瘤细胞侵入通过细胞外基质（ECM）的能力，是在转移表型1-3的一个标志。入侵室已被广泛用作体外的工具来研究癌细胞的侵袭和转移，并可以提供知识， 体内肿瘤的侵袭和转移的机制。 腔室包括嵌套细胞培养板的孔中的圆柱形的细胞培养插入物。插入件的底部是限定孔径的半透聚碳酸酯或聚苯乙烯膜。 在标准侵袭实验，将细胞接种到在插入膜用无血清培养基，并置于细胞培养孔被填充有血清或血清样化学引诱物送等了一个趋化的力量。移动至该力驱动细胞通过的半透膜（迁移）或可替代地，通过细胞外基质（侵入），然后可以使用常规的染料检测和定量细胞数染色的涂层。细胞数，然后根据迁移和侵袭的非侵入性的细胞系的范围内归一化。此方法允许研究者评估在各种条件，包括遗传操作不同类型的细胞的侵袭性潜力。 激素，细胞因子和生长因子是血清的关键组成部分，并越来越多地被示出为与驱动侵袭性表型4相关联。然而，在自然界，角色，和特异性这些趋化血清分子仍然介导入侵仍然无法实现。我们面临的挑战仍然是确定哪些具体因素血清或血清样趋化因子负责驾驶癌细胞侵袭以及什么分子可以抑制入侵过程。在这份报告中，我们描述了一种方法来评估潜在激素，细胞因子和/或血清中存在的生长因子的参与，如分子找到癌细胞的趋化和侵袭。 在此提出的改性协议，木炭剥离用于去除激素，细胞因子和生长因子，由2％胎牛血清（FBS），以产生2％的活性炭脱色的FBS（CS-FBS）。两个2％FBS和2％CS-FBS的被用作试剂来建立趋化力来驱动癌细胞侵袭。用2％CS-FBS的作为相比于正常的2％FBS的趋化剂能提供若干好处，包括第一，最突出的是，以研究在减少/相对缺乏激素，细胞因子和生长因子的癌症侵袭的表型的能力。它还使研究者评估是否集体去除激素，生长因子，以及CYtokines引起侵袭性指数的增加或减少。该测定然后设计，以确定是否增加一个单独的组件，被指定为"X"，可以抑制，减少，增加，或侵入索引恢复到原来的值（参见图2和3）。虽然这种方法是具体用于实现生理水平是从血清中木炭提除去的组件，还应当注意的是，木炭汽提也可以影响信号转导途径。例如，已经报道，木炭剥离能降低骨祖细胞的碱性磷酸酶活性，并通过减少MAPK活化剂5的诱导脂肪生成。木炭剥离介质是可商购，并且基于协议概述，结合木炭与葡聚糖和孵育胎牛血清O / N 6。 该测定是在respo开发NSE到报告的Zucker 等人 7的结果的作者证明朝向媒体与正常的2％FBS的迁移时的突变体的表型影响间隙连接通讯证明增加的侵袭索引。这一增长与活性炭处理血清替代被淘汰。从该结果中，作者得出结论认为，这种突变影响朝向亲脂性分子迁移（更具体地，激素，生长因子，细胞因子或）7。它公知的是激素，生长因子和细胞因子介导的信号参与肿瘤促进和侵入4通路。因此，对于科学调查，以确定哪些因子（S）的驱动的肿瘤侵袭其特定的癌细胞或突变体的研究之中是重要的。该试验的目的是为解决在其生理浓度各组分的作用，因为，根据该成分的浓度之间的差来确定与在正常的FBS和CS-FBS的X";＃8220。通过此法的发展，研究人员可以更深入地了解具体的侵入途径管理他们的系统。 激素，生长因子，和细胞因子经常被归类为促癌8。其中的一些因素，如EGF被用作化学引诱物中浸润室9直接来源。因此，它很可能是这些代表了血清肿瘤直接侵犯的主要组成部分。该协议提出了一种简单的，但显著，修改现有的体外侵袭测定法，其允许研究者评估在介导癌细胞的侵袭性潜力激素，生长因子和细胞因子的参与。但是，在测定的目的是提供一个答案的研究者以过程是否将是有效的对他们的研究在分析的早期步骤，以免使用宝贵的时间而r物资跟不上，如果认为没有必要。此方法使用的CS-FBS和依赖于研究者酌情决定哪个分子追求铅候选潜在介导癌细胞侵袭。从这些分析的结果应该被证明在确定其中血清组分充当被研究的化学引诱物或抑制剂对特定细胞系或突变体是有用的。此外，这种方法可以帮助研究者确定主要的信号通路或者促进或抑制癌细胞侵袭;从而引导未来的药物设计。

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		<tr height="22">
			<td height="22" style="height:22px;width:367px;">Item</td>
			<td style="width:195px;">Vendor</td>
			<td style="width:136px;">Catalog #</td>
			<td style="width:289px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td>BioCoat Control Inserts with 8.0&#181;m PET Membrane 
			(12 in each of two 24-Well Plates)</td>
			<td>Corning</td>
			<td>354578</td>
			<td>polyethylene teraphthalate (PET) membranes&#160;&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Collagen I, rat tail</td>
			<td>Corning</td>
			<td>354236</td>
			<td>Source = rat tail tendon, purity = 90%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Fixative; Diff-Quick</td>
			<td>Thermo Fisher Scientific</td>
			<td>NC9844047</td>
			<td>Methanol-based fixative, Hmatoxylin/Eosin</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Fetal bovine Serum</td>
			<td>Life Technologies</td>
			<td>16000</td>
			<td>Designated as FBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Charcoal Stripped Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>12676</td>
			<td>Designated as CS-FBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Fetal bovine Serum</td>
			<td>Thermo Scientific Hyclone</td>
			<td>SH30070</td>
			<td>Designated as FBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Charcoal Stripped Fetal Bovine Serum</td>
			<td>Thermo Scientific Hyclone</td>
			<td>SH30068</td>
			<td>Designated as CS-FBS</td>
		</tr>
	</tbody>
</table>

a modified in vitro invasion assay to determine the potential role of hormones, cytokines and/or growth factors in mediating cancer cell invasion

血清作为趋化对其中的癌细胞迁移和侵袭，促进他们的血管内进入微血管。然而，实际的分子朝向该细胞迁移仍然无法实现。本变形侵袭实验已经开发，以确定哪一个驱动器细胞迁移和侵袭的目标。这种技术比较下三个条件的侵袭索引，以确定是否一个特定的激素，生长因子，细胞因子或在介导癌细胞的侵袭性潜力的作用。这些条件包括:i）正常牛胎儿血清（FBS），ⅱ）活性炭处理的FBS（CS-FBS）的功能，从而激素，生长因子，以及细胞因子和iii）的CS-FBS +分子（表示为"X"）。相比FBS细胞侵袭与CS-FBS的显著的变化，表明激素，细胞因子和生长因子介导的变化的参与。各个分子然后可以重新添加到CS-FBS以测定他们的重新能力诗句或抢救入侵表型。此外，两个或更多的因素可以结合，以评估该添加剂或多种分子在驾驶或抑制侵袭协同效应。总体而言，该方法能够使研究者确定是否激素，细胞因子，和/或生长因子通过作为化学引诱物或侵入的抑制剂为特定类型的癌细胞的或特定的突变体在细胞侵袭的作用。通过识别特定的趋化因子和抑制剂，这个修改后的入侵检测可能有助于阐明信号通路，直接癌细胞侵袭。

入侵指数是根据归一化，以一种非侵入性的细胞系计算每个条件。对于我们的实验中，我们使用1205LU黑素瘤细胞系，建立了变体的稳定细胞株作为我们的侵入线以及癌前无创变型中，WM793从中1205LU细胞衍生10用作一个逻辑控制。我们还利用I型胶原作为基质侵入，因为这是真皮的主要成分。这是根据先前的研究，从而最优侵袭基质变化的基础上的细胞系和一致性的与体内的程度的结果...

Watch this Scientific Journal Video about A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion at JoVE.com

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 ...

a-modified-vitro-invasion-assay-to-determine-potential-role-hormones

Research

JoVE Journal

Medicine

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

10.6K 次观看.  Roswell Park Cancer Institute. 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.

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

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

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

一个<em在体外系统来研究肿瘤休眠和转移性增长

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

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 ...

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 ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

建立一个人类多发性骨髓瘤异种移植模型中的鸡，研究肿瘤的生长，侵袭和血管生成

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

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 ...

一种改进

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