Name: live-imaging of breast epithelial cell migration after the transient depletion of tip60
Uploaded: 2017-12-07T13:00:00.000+00:00
Duration: 8 min 13 s
Description: Watch this Scientific Journal Video about Live-imaging of Breast Epithelial Cell Migration After the Transient Depletion of TIP60 at JoVE.com

我们感谢贾实验室的成员的有益的讨论和评论。s.j 得到了新加坡国家研究基金会和新加坡教育部在其卓越研究中心倡议下向新加坡癌症科学研究所 (R-713-006-014-271) 提供的赠款, 国家医学研究理事会 ( CBRG - ;BNIG11nov001 和 CS-表意文字;R-713-000-162-511), 教育部学术研究基金 (教育部 AcRF 级 1 T1-2012 10月04日)。Y.Z.、分析和 C.Y.T. 都得到新加坡国立大学新加坡癌症科学研究所颁发的研究生奖学金的支持。

1. 准备
<ol><li>准备 MCF10A 培养基: Dulbecco 氏改良鹰培养基 (DMEM)/F12 (1:1) 与5% 马血清, 20 ng/毫升上皮生长因子, 0.5 毫克/毫升的可的松, 100 ng/毫升霍乱毒素, 和10µg/毫升胰岛素。</li>
	<li>制备无血清培养基: DMEM/F12 (1:1), 20 ng/毫升上皮生长因子, 0.5 毫克/毫升可的松, 100 ng/毫升霍乱毒素, 和10µg/毫升胰岛素。</li>
	<li>使用以下 siRNA 序列: siControl: 转发: 5 "-CGUACGCGGAAUACUUCGAdTdT-3" 反向: 5 "-UCGAAGUAUUCCGCGUACGdTdT-3" siTIP60: 转发: 5 "-UGAUCGAGUUCAGCUAUGAdTdT-3" 反向: 5 "-UCAUAGCUGAACUCGAUCAdTdT-3"</li>
	<li>使用10厘米细胞培养皿和24孔板。</li>
</ol>2. 使用 siRNA 的瞬态损耗 TIP60
<ol><li>在1天, 分15µL 转染试剂 (例如,体 RNAiMAX) 和2毫升的还原血清培养基 (例如,优化-记忆), 并将它们与每µL (10 siRNA 库存) 的10µM 分别混合。</li>
	<li>室温下孵育混合物20分钟。</li>
	<li>种子 1 x 106 MCF10A 细胞连同3毫升培养基在 10 cm 盘中。使用例计数单元格。</li>
	<li>加入 siRNA 混合物滴到10厘米的盘子里。</li>
	<li>摇动盘子, 确保细胞在放入37° c 恒温箱前均匀分布。</li>
	<li>经过6小时的孵化, 用8毫升的新鲜培养基取代含 siRNA 的培养基。</li>
	<li>2天, 准备另一批 siRNA 混合物, 在室温下孵育20分钟。</li>
	<li>抽出8毫升的培养基添加在1天, 代之以3毫升的新鲜培养基。</li>
	<li>将第二批 siRNA 的混合物放到盘子里滴下。摇动碗碟, 确保试剂在放入孵化箱前完全混合。</li>
	<li>6 h 后, 用8毫升的新鲜培养基取代含 siRNA 的培养基。</li>
</ol>3. 伤口愈合试验用种子细胞
<ol><li>在3天, 丢弃培养基, 用2毫升磷酸缓冲盐 (PBS) 缓冲液冲洗细胞一次。丢弃 PBS 缓冲区。</li>
	<li>处理 siControl 和 siTIP60-treated 细胞, 加入1毫升的1x 胰蛋白酶-EDTA 缓冲到板块。把它放回孵化器20分钟。</li>
	<li>添加4毫升的完整培养基到每个板块, 重细胞的移, 并转移到一个15毫升的管。</li>
	<li>离心机在200克为5分钟, 并删除上清液。</li>
	<li>用5毫升无血清培养基重新悬浮细胞, 用例计数细胞。</li>
	<li>为 siControl 和 siTIP60 准备一个 6 x 105细胞/毫升的细胞悬浮液;确保细胞混合良好。将500µL 的细胞悬浮液添加到24孔板的一个井中, 以确保 100% 70-100。种子5至6口井分别为 siControl 和 siTIP60。</li>
	<li>轻轻摇晃板来回, 然后侧面, 以确保均匀分配。避免圆周运动。把盘子放在孵化箱里24小时。</li>
	<li>收获剩余的细胞以检查 TIP60 的击倒效率。为此, 将剩余的细胞悬浮在200克, 5 分钟, 除去上清液。</li>
	<li>使用商业试剂分离 RNA, 按照制造商的协议, 并进行实时定量 PCR 评估击倒效率。</li>
</ol>4. 创建伤口
<ol><li>在相衬显微镜下检查, 确保细胞完全附着。观察显微镜下的细胞 (4X、10X 和20X 的放大倍数), 以确保细胞完全附着, 形成一个汇合的单层细胞。 注: siControl 细胞通常比 siTIP60 细胞附着更好。这是意料之中的, 因为 TIP60 的耗尽使细胞的间质更像20。在这里, 使用正常的台式白光相衬显微镜。</li>
	<li>要产生伤口, 用200µL 吸管尖端轻轻划伤一条穿过井中心的直线;所产生的伤口长度与井径相同。对余下的水井重复这一步。每井使用一个新的吸管尖端。</li>
	<li>用无血清培养基轻轻冲洗每一个井5次, 去除分离的细胞。</li>
	<li>每井加1毫升无血清培养基, 并进行活体细胞成像。</li>
</ol>5. 活细胞成像的设置
<ol><li>在设置活细胞成像之前, 打开显微镜和温度控制至少1小时, 以确保燃烧室内的温度达到37° c。</li>
	<li>在开始活细胞成像之前, 打开二氧化碳 (CO2) 气体供应。将 CO2浓度设置为5%。</li>
	<li>将24井板放在显微镜的舞台上, 选择物镜, 10X 放大成像。将发射的光直接放到目镜而不是相机上, 调整焦点以定位伤口和周围的细胞。 注: 在这种情况下, 使用了相衬的白光。</li>
	<li>使用活细胞观察系统来标记伤口在每个井中的位置。 注: 活细胞成像系统的自动化阶段允许在多个位置进行图像采集。每隔一段时间就会对每个标记位置进行图像处理。</li>
	<li>设置时间条件: 1 小时间隔和72小时;根据这个设置, 系统将每小时在每一个标记的位置上拍照 72 h. 设置任何所需的时间间隔和监视期间。 注意: 在这里, 72 h 的持续时间是最好的, 因为这些细胞在无血清培养基中72小时后开始死亡。</li>
</ol>6. 跟踪单细胞移动
<ol><li>在72小时后导出数据. 将数据同时导出为 JPEG 格式的图像和 AVI 格式的视频。 注意: 这很重要, 因为 ImageJ 软件只能识别 AVI 格式的视频。siTIP60 和 siControl 细胞之间的不同细胞迁移速度可以在视频中清晰地观察到。</li>
	<li>在 ImageJ 软件中打开 AVI 视频。</li>
	<li>打开 MTrackJ 插件 (必须单独下载和安装插件): 插件 > MTrackJ。</li>
	<li>通过单击 MtrackJ 工具栏中的 "添加" 选项卡, 为从视频中选择的单个单元格添加音轨。在侵入前的尖端选择一个细胞, 一个可以自行迁移的细胞 (单细胞迁移)。跟踪视频中所选单元格的移动并使用 MtrackJ 跟踪它们。</li>
	<li>通过单击工具栏上的 "测量" 选项卡来测量移动距离。 注: 在这里, 几乎没有细胞在 siControl 移动作为单细胞, 使它难以测量单细胞移动, 而许多 siTIP60 细胞移动作为单细胞。然而, 位于侵入前沿的细胞的运动仍可在 siControl 和 siTIP60 细胞中追踪和计算。</li>
	<li>用 AVI 格式的曲目保存视频。</li>
</ol>7. 细胞迁移能力的量化
<ol><li>对于量化, 选择图像在0小时和图像在 "n" h, 其中 "n" 指的 timepoint 时, 要么 siTIP60 或 siControl 细胞完全封闭的伤口。 注: 在这里, 48 h 被选中, 因为 siTIP60 细胞的伤口几乎完全闭合。</li>
	<li>使用以下公式对使用 ImageJ 的迁移百分比进行量化: (在 "n" h 的伤口面积 0 h 区)/伤口面积0ĥ x 100。</li>
</ol>

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作者声明他们没有竞争的金融利益。

众所周知, 在细胞迁移的过程中, 细胞可以以附着板的形式移动;松散簇;或单个独立的单元格。细胞侵袭的模式和动力可以从一个条件变化到另一个, 而表型在数小时内就会改变。传统的伤口愈合试验是基于从显微镜拍摄的长时间间隔, 如12或24小时的快照照片。这使得难以捕捉伤口闭合的动态和细胞运动的模式。
另一方面, 活细胞成像系统监测细胞迁移在一个更短的时间间隔 (30...

HIV-达互动蛋白 60 kDa (TIP60) 是一种赖氨酸乙酰, 可以乙酰组蛋白和非组蛋白蛋白质1,2。它的功能被发现具有多重信号通路的含义, 包括转录, DNA 损伤修复, 和凋亡 1,3,4,5,67,8. 此外, TIP60 常 downregulated 于癌症, 其下调与癌症转移和生存率差有关9、10、11、12, 13,14。肿瘤转移是癌症相关死亡的主要原因。它是一个多步过程, 转移的初始步骤涉及肿瘤细胞向相邻组织的迁移和侵袭15,16。为了做到这一点, 肿瘤细胞首先必须脱离原发肿瘤肿块, 要么以集体侵入细胞表的形式, 要么作为分离的单个细胞17。在这个过程中, 肿瘤细胞经常发生上皮间转移 (急诊), 导致形态学和细胞黏附能力的变化17,18。
对肿瘤细胞的迁移和侵袭能力进行了研究, 并对其进行了体外培养。其中, 伤口愈合试验是最有效和经济的19。首先, 这种方法涉及在细胞的汇合单层上创建人工间隙, 从而允许细胞迁移和闭合间隙。第二, 在实验开始和结束时可以捕捉到间隙的图像。最后, 通过比较间隙闭合来确定细胞迁移率。相衬显微镜通常用于捕捉图像的传统伤口愈合的化验。伤口愈合试验的另一个优点是它部分类似于体内肿瘤细胞的迁移。与体内肿瘤转移相似, 伤口愈合过程中的细胞迁移表现为上皮片的集体迁移和分离单细胞的迁移。
然而, 细胞迁移是已知的动态的, 并且有需要记录细胞的移动在实时。例如, 一个传统的伤口愈合试验不允许研究员分析单细胞运动。另一方面, 培养伤口愈合的细胞通常是缺乏血清的, 以抑制细胞增殖。这样做是为了排除因细胞增殖而导致间隙闭合的可能性。尽管如此, 还是会有一些增殖和细胞死亡, 并且在实验前后所拍摄的相衬图像无法区分它们。
活细胞成像技术解决了这一局限性的常规伤口愈合的化验。使用实时成像显微镜结合 CO2孵化器和适当的温度控制, 研究人员可以测量间隙大小及其在一段时间内的闭合速率, 同时跟踪位于侵入前线因此, 实施活细胞成像监测细胞迁移不仅将提供更好的可视化细胞迁移, 但也将允许区分细胞迁移和细胞增殖的可能性, 从而提供了一个更细胞迁移的可靠分析。
在这项研究中, MCF10A 乳腺上皮细胞被用来证明伤口愈合试验和活细胞成像的结合, 以研究 TIP60 在细胞迁移中的作用。TIP60 的耗竭导致 MCF10A 细胞迁移能力的提高。

<table><tbody><tr><td>MCF10A cell</td><td>ATCC</td><td>CRL-10317TM</td><td>Breast epithelial cell</td></tr><tr><td>DMEM/F12 media (1:1)</td><td>Gibco</td><td>11330-032</td><td>MCF10A culture media</td></tr><tr><td>Epithelial growth factor (EGF)</td><td>Peprotech</td><td>AF-100-15</td><td>Supplements for MCF10A culture media</td></tr><tr><td>Cholera Toxin</td><td>Sigma-Aldrich</td><td>C-8052</td><td>Supplements for MCF10A culture media</td></tr><tr><td>Hydrocortisone</td><td>Sigma-Aldrich</td><td>H-0888</td><td>Supplements for MCF10A culture media</td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>I-1882</td><td>Supplements for MCF10A culture media</td></tr><tr><td>House serum</td><td>Gibco</td><td>16050-122</td><td>Supplements for MCF10A culture media</td></tr><tr><td>Trypsin</td><td>Gibco</td><td>25200-056</td><td>For MCF10A detach from plate</td></tr><tr><td>Hemacytometer</td><td>Fisher Scientific</td><td>267110</td><td>For counting cell</td></tr><tr><td>Opti-MEM reduced serum media</td><td>Gibco</td><td>31985-070</td><td>For siRNA mixture</td></tr><tr><td>Lipofectamine RNAiMAX</td><td>Invitrogen/Life Technologies</td><td>56532</td><td>Transfect reagent</td></tr><tr><td>Trizol reagent</td><td>Invitrogen/Life Technologies</td><td>15596-026</td><td>For mRNA extraction</td></tr><tr><td>iScript cDNA Synthesis Kit</td><td>Bio-Rad</td><td>170-8891</td><td>For cDNA generation</td></tr><tr><td>iTaq Universal SYBR Green Supermix</td><td>Bio-Rad</td><td>172-5125</td><td>For real time qPCR</td></tr><tr><td>7500 Fast Real Time PCR system</td><td>Biosystems</td><td></td><td>Real time qPCR mechine</td></tr><tr><td>10-cm dish</td><td>Greiner bio-one</td><td>664160</td><td>For Knockdown experiment</td></tr><tr><td>24-well plate</td><td>Cellstar</td><td>662160</td><td>For wound healing assay</td></tr><tr><td>siControl siRNA</td><td>Self designed</td><td>Self designed</td><td>CGUACGCGGAAUACUUCGAdTdT</td></tr><tr><td>siTIP60 siRNA</td><td>Self designed</td><td>Self designed</td><td>UGAUCGAGUUCAGCUAUGAdTdT</td></tr><tr><td>Live cell observer</td><td>Zeiss</td><td></td><td>Zeiss inverted Cell Observer for live cell experiments</td></tr></tbody></table>

live-imaging of breast epithelial cell migration after the transient depletion of tip60

伤口愈合试验是有效的, 也是研究细胞迁移的最经济的方法之一体外。传统上, 图像是在实验开始和结束时使用相衬显微镜, 并通过闭合伤口来评估细胞的迁移能力。然而, 细胞运动是一种动态现象, 传统的方法不允许追踪单细胞运动。为了改善目前的伤口愈合检测方法, 我们使用活细胞成像技术实时监测细胞迁移。这种方法使我们能够确定细胞迁移率的基础上的细胞跟踪系统, 并提供了更明确的区分细胞迁移和细胞增殖。在这里, 我们演示了使用活细胞成像在伤口愈合的检测, 以研究不同的迁移能力的影响乳腺上皮细胞 TIP60 的存在。由于细胞运动是高度动态的, 我们的方法提供了更多的洞察力愈合的过程, 而不是一个伤口闭合拍摄的传统成像技术用于伤口愈合化验。

基于活细胞成像的细胞迁移试验的一般图式 图 1A是此协议的架构。MCF10A 细胞转染 siControl 显示典型的上皮形态学。在 TIP60 耗尽后, MCF10A 细胞与对照细胞相比更具间质性 (图 1B)。
TIP60 击倒效率的检验 图 2显示了 TIP60 击倒效率。siTI...

Watch this Scientific Journal Video about Live-imaging of Breast Epithelial Cell Migration After the Transient Depletion of TIP60 at JoVE.com

Live-imaging of Breast Epithelial Cell Migration After the Transient Depletion of TIP60

TIP60 短暂耗竭后乳腺上皮细胞迁移的活体影像学研究

在这里, 我们提出了实时监测细胞迁移在伤口愈合试验中使用 TIP60-depleted MCF10A 乳腺上皮细胞。在我们的协议中实现了活细胞成像技术, 使我们能够实时和跨时间地分析和可视化单细胞运动。

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the ...

live-imaging-breast-epithelial-cell-migration-after-transient

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

6.8K 次观看.  National University of Singapore. 在这里, 我们提出了实时监测细胞迁移在伤口愈合试验中使用 TIP60-depleted MCF10A 乳腺上皮细胞。在我们的协议中实现了活细胞成像技术, 使我们能够实时和跨时间地分析和可视化单细胞运动。

视频: Live-imaging of Breast Epithelial Cell Migration After the Transient Depletion of TIP60

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS expression system1 in zebrafish larvae, and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells. The KalTA4-ERT2/UAS system is both inducible and reversible which allows us to induce cell transformation with precise temporal/spatial resolution in vivo. This provides us with a unique opportunity to live image how individual preneoplastic cells interact with host tissues as soon as they emerge, then follow their progression as well as regression. Recent studies in zebrafish larvae have shown a trophic function of innate immunity in the earliest stages of tumorigenesis2,3. Our inducible system would allow us to live image the onset of cellular transformation and the subsequent host response, which may lead to important insights on the underlying mechanisms for the regulation of oncogenic trophic inflammatory responses. We also discuss how one might adapt our protocol to achieve temporal and spatial control of ectopic gene expression in any tissue of interest.

Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells in zebrafish larvae.

先天免疫和癌前细胞间的相互作用使用可诱导的Gal4 / UAS表达系统在斑马鱼幼体皮肤实时成像

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS ...

科研

发育生物学

Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane resistance in cancerous cells drastically reduces the effectiveness of the drugs' long-term usage. Accumulated evidence suggests that the mechanisms underlying taxane resistance include both general mechanisms, such as the development of multidrug resistance due to the overexpression of drug-efflux proteins, and taxane-specific mechanisms, such as those that involve microtubule dynamics. 
Because taxanes target cell microtubules, measuring microtubule dynamic instability is an important step in determining the mechanisms of taxane resistance and provides insight into how to overcome this resistance. In the experiment, an in vivo method was used to measure microtubule dynamic instability. GFP-tagged &#945;-tubulin was expressed and incorporated into microtubules in MCF-7 cells, allowing for the recording of the microtubule dynamics by time lapse using a sensitive camera. The results showed that, as opposed to the non-resistant parental MCF-7CC cells, the microtubule dynamics of docetaxel-resistant MCF-7TXT cells are insensitive to docetaxel treatment, which causes the resistance to docetaxel-induced mitotic arrest and apoptosis. This paper will outline this in vivo method of measuring microtubule dynamic instability.

In this paper, we report a protocol describing an in vivo method to measure microtubule dynamic instability in docetaxel-resistant breast cancer cells (MCF-7TXT). In this method, a deconvolution microscopy imaging system is used to detect the expression of GFP-tubulin in target cells.

实时成像来研究抗紫杉烷乳腺癌微管动态失稳

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane ...

癌症研究

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

活细胞成像的移植细胞在一个三维矩阵表示荧光标记的蛋白质

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

生物工程

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

分析<em&gt;在体内</em&gt;细胞迁移使用细胞移植和时间推移成像在斑马鱼胚胎

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

癌症干细胞迁移的使用进行划分的微流控芯片和活细胞成像的评价

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

医学

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

活体细胞成像技术研究胶质瘤脑干细胞的迁移和侵袭

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

使用自动活单元成像仪的简单迁移/入侵工作流

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

延时成像的主要癌前乳腺上皮细胞来源于遗传工程小鼠乳腺癌模型

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Isolation and Time-Lapse Imaging of Primary Mouse Embryonic Palatal Mesenchyme Cells to Analyze Collective Movement Attributes

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and fusion above the tongue. Defects in this process lead to cleft palate, a common birth defect. Recent studies have shown that palatal shelf elevation involves a remodeling process that transforms the orientation of the shelf from a vertical to a horizontal one. The role of the palatal shelf mesenchymal cells in this dynamic remodeling has been difficult to study. Time-lapse-imaging-based quantitative analysis has been recently used to show that primary mouse embryonic palatal mesenchymal (MEPM) cells can self-organize into a collective movement. Quantitative analyses could identify differences in mutant MEPM cells from a mouse model with palate elevation defects. This paper describes methods to isolate and culture MEPM cells from E13.5 embryos-specifically for time-lapse imaging-and to determine various cellular attributes of collective movement, including measures for stream formation, shape alignment, and persistence of direction. It posits that MEPM cells can serve as a proxy model for studying the role of palatal shelf mesenchyme during the dynamic process of elevation. These quantitative methods will allow investigators in the craniofacial field to assess and compare collective movement attributes in control and mutant cells, which will augment the understanding of mesenchymal remodeling during palatal shelf elevation. Furthermore, MEPM cells provide a rare mesenchymal cell model for investigation of collective cell movement in general.

We present a protocol for isolation and culture of primary mouse embryonic palatal mesenchymal cells for time-lapse imaging of two-dimensional (2D) growth and wound-repair assays. We also provide the methodology for analysis of the time-lapse imaging data to determine cell-stream formation and directional motility.

原代小鼠胚胎腭间充质细胞的分离和延时成像，以分析集体运动属性

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

随机使用时移视频显微镜细胞迁移的定量分析

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...