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 border="0" cellpadding="0" cellspacing="0" width="874">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">MCF10A cell</td>
			<td style="width:211px;">ATCC</td>
			<td style="width:119px;">CRL-10317TM</td>
			<td style="width:257px;">Breast epithelial cell</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">DMEM/F12 media (1:1)</td>
			<td style="width:211px;">Gibco</td>
			<td style="width:119px;">11330-032</td>
			<td>MCF10A culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Epithelial growth factor (EGF)</td>
			<td style="width:211px;">Peprotech</td>
			<td style="width:119px;">AF-100-15</td>
			<td>Supplements for MCF10A culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Cholera Toxin</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">C-8052</td>
			<td>Supplements for MCF10A culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Hydrocortisone</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">H-0888</td>
			<td>Supplements for MCF10A culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Insulin</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">I-1882</td>
			<td>Supplements for MCF10A culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">House serum</td>
			<td style="width:211px;">Gibco</td>
			<td style="width:119px;">16050-122</td>
			<td>Supplements for MCF10A culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Trypsin</td>
			<td style="width:211px;">Gibco</td>
			<td style="width:119px;">25200-056</td>
			<td>For MCF10A detach from plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Hemacytometer</td>
			<td style="width:211px;">Fisher Scientific</td>
			<td style="width:119px;">267110</td>
			<td>For counting cell</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Opti-MEM reduced serum media</td>
			<td style="width:211px;">Gibco</td>
			<td style="width:119px;">31985-070</td>
			<td>For siRNA mixture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Lipofectamine RNAiMAX</td>
			<td style="width:211px;">Invitrogen/Life Technologies</td>
			<td style="width:119px;">56532</td>
			<td>Transfect reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Trizol reagent</td>
			<td style="width:211px;">Invitrogen/Life Technologies</td>
			<td style="width:119px;">15596-026</td>
			<td>For mRNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">iScript cDNA Synthesis Kit</td>
			<td style="width:211px;">Bio-Rad</td>
			<td style="width:119px;">170-8891</td>
			<td>For cDNA generation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">iTaq Universal SYBR Green Supermix</td>
			<td style="width:211px;">Bio-Rad</td>
			<td style="width:119px;">172-5125</td>
			<td>For real time qPCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">7500 Fast Real Time PCR system</td>
			<td style="width:211px;">Biosystems</td>
			<td style="width:119px;"></td>
			<td>Real time qPCR mechine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">10-cm dish</td>
			<td style="width:211px;">Greiner bio-one</td>
			<td style="width:119px;">664160</td>
			<td>For Knockdown experiment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">24-well plate</td>
			<td style="width:211px;">Cellstar</td>
			<td style="width:119px;">662160</td>
			<td>For wound healing assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">siControl siRNA</td>
			<td style="width:211px;">Self designed</td>
			<td style="width:119px;">Self designed</td>
			<td>CGUACGCGGAAUACUUCGAdTdT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">siTIP60 siRNA</td>
			<td style="width:211px;">Self designed</td>
			<td style="width:119px;">Self designed</td>
			<td>UGAUCGAGUUCAGCUAUGAdTdT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Live cell observer</td>
			<td style="width:211px;">Zeiss</td>
			<td style="width:119px;"></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

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

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

利用功能基因组筛选识别在肿瘤细胞球体培养潜在的新药物靶标

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

科研

癌症研究

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

门静脉注射模型来研究肝转移乳腺癌

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

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 ...

Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying cancer metastasis in vivo, other efficient, reliable, low-cost models are needed to quickly access the potential effects of (epi)genetic changes or pharmacological compounds. As such, we illustrate and explain the feasibility of xenograft models using human breast cancer cells injected into zebrafish embryos to support this goal. Under the microscope, fluorescent proteins or chemically labeled human breast cancer cells are transplanted into transgenic zebrafish embryos, Tg (fli:EGFP), at the perivitelline space or duct of Cuvier (Doc) 48 h after fertilization. Shortly afterwards, the temporal-spatial process of cancer cell invasion, dissemination, and metastasis in the living fish body is visualized under a fluorescent microscope. The models using different injection sites, i.e., perivitelline space or Doc are complementary to one another, reflecting the early stage (intravasation step) and late stage (extravasation step) of the multistep metastatic cascade of events. Moreover, peritumoral and intratumoral angiogenesis can be observed with the injection into the perivitelline space. The entire experimental period is no more than 8 days. These two models combine cell labeling, micro-transplantation, and fluorescence imaging techniques, enabling the rapid evaluation of cancer metastasis in response to genetic and pharmacological manipulations.

Here, we describe xenograft zebrafish models using two different injection sites, i.e., perivitelline space and duct of Cuvier, to investigate the invasive behavior and to assess the intravasation and extravasation potential of human breast cancer cells, respectively.

在斑马鱼胚胎人乳腺癌细胞的侵袭行为

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying ...

Using the Dot Assay to Analyze Migration of Cell Sheets

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move within tissues in a tightly orchestrated manner. During tumor development, this equilibrium is disturbed, and tumor cells leave the epithelium of origin to invade the local microenvironment, to travel to distant sites, and to ultimately form metastatic tumors at distant sites. The dot assay is a simple, two-dimensional unconstrained migration assay, to assess the net movement of cell sheets into a cell-free area, and to analyze parameters of cell migration using time-lapse imaging. Here, the dot assay is demonstrated using a human invasive, lung colony forming breast cancer cell line, MCF10CA1a, to analyze the cells' migratory response to epidermal growth factor (EGF), which is known to increase malignant potential of breast cancer cells and to alter the migratory phenotype of cells.

Cell migration is essential for development, tissue maintenance and repair, and tumorigenesis, and is regulated by growth factors, chemokines, and cytokines. This protocol describes the dot assay, a two-dimensional, unconstrained migration assay to assess the migratory phenotype of attached, cohesive cell sheets in response to microenvironmental cues.

用点法分析细胞板的迁移

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move ...

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 ...

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon named apoptosis reversal leads to increased tumorigenicity in various cell models under different stimuli. Previous studies have been focused on tracking this process in vitro and in vivo; however, the isolation of real reversed cells has yet to be achieved, which limits our understanding on the consequences of apoptosis reversal. Here, we take advantage of a Caspase-3/7 Green Detection dye to label cells with activated caspases after apoptotic induction. Cells with positive signals are further sorted out by fluorescence-activated cell sorting (FACS) for recovery. Morphological examination under confocal microscopy helps confirm the apoptotic status before FACS. An increase in tumorigenicity can often be attributed to the elevation in the percentage of cancer stem cell (CSC)-like cells. Also, given the heterogeneity of breast cancer, identifying the origin of these CSC-like cells would be critical to cancer treatment. Thus, we prepare breast non-stem cancer cells before triggering apoptosis, isolating caspase-activated cells and performing the apoptosis reversal procedure. Flow cytometry analysis reveals that breast CSC-like cells re-appear in the reversed group, indicating breast CSC-like cells are transited from breast non-stem cancer cells during apoptosis reversal. In summary, this protocol includes the isolation of apoptotic breast cancer cells and detection of changes in CSC percentage in reversed cells by flow cytometry.

Here, we present a protocol to isolate apoptotic breast cancer cells by fluorescence-activated cell sorting and further detect the transition of breast non-stem cancer cells to breast cancer stem cell-like cells after apoptosis reversal by flow cytometry.

细胞凋亡逆转后新形成乳腺癌干细胞样细胞的流式细胞仪检测

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon ...

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction of differentiation of two mouse breast epithelial cell lines, HC11 and EpH4, and their use to study complementary stages of mammary gland development and neoplastic transformation.
The HC11 mouse breast epithelial cell line originated from the mammary gland of a pregnant Balb/c mouse. It differentiates when grown to confluence attached to a plastic Petri dish surface in medium containing fetal calf serum and Hydrocortisone, Insulin and Prolactin (HIP medium). Under these conditions, HC11 cells produce the milk proteins &#946;-casein and whey acidic protein (WAP), similar to lactating mammary epithelial cells, and form rudimentary mammary gland-like structures termed &#34;domes&#34;.
The EpH4 cell line was derived from spontaneously immortalized mouse mammary gland epithelial cells isolated from a pregnant Balb/c mouse. Unlike HC11, EpH4 cells can fully differentiate into spheroids (also called mammospheres) when cultured under three-dimensional (3D) growth conditions in HIP medium. Cells are trypsinized, suspended in a 20% matrix consisting of a mixture of extracellular matrix proteins produced by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, plated on top of a layer of concentrated matrix coating a plastic Petri dish or multiwell plate, and covered with a layer of 10% matrix-containing HIP medium. Under these conditions, EpH4 cells form hollow spheroids that exhibit apical-basal polarity, a hollow lumen, and produce &#946;-casein and WAP.
Using these techniques, our results demonstrated that the intensity of the cadherin/Rac signal is critical for the differentiation of HC11 cells. While Rac1 is necessary for differentiation and low levels of activated RacV12 increase differentiation, high RacV12 levels block differentiation while inducing neoplasia. In contrast, EpH4 cells represent an earlier stage in mammary epithelial differentiation, which is inhibited by even low levels of RacV12.

We describe techniques for differentiation induction of two breast epithelial lines, HC11 and EpH4. While both require fetal calf serum, insulin, and prolactin to produce milk proteins, EpH4 cells can fully differentiate into mammospheres in three-dimensional culture. These complementary models are useful for signal transduction studies of differentiation and neoplasia.

小鼠乳腺皮下HC11和EpH4细胞的分化

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction ...

Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation capacity of mouse prostate basal and luminal cells during development, tissue-regeneration and transformation. However, evaluating cell-intrinsic and extrinsic regulators of prostate epithelial differentiation capacity using a lineage tracing approach often requires extensive breeding and can be cost-prohibitive. In the prostate organoid assay, basal and luminal cells generate prostatic epithelium ex vivo. Importantly, primary epithelial cells can be isolated from mice of any genetic background or mice treated with any number of small molecules prior to, or after, plating into three-dimensional (3D) culture. Sufficient material for evaluation of differentiation capacity is generated after 7-10 days. Collection of basal-derived and luminal-derived organoids for (1) protein analysis by Western blot and (2) immunohistochemical analysis of intact organoids by whole-mount confocal microscopy enables researchers to evaluate the ex vivo differentiation capacity of prostate epithelial cells. When used in combination, these two approaches provide complementary information about the differentiation capacity of prostate basal and luminal cells in response to genetic or pharmacological manipulation.

Mouse prostate organoids represent a promising context to evaluate mechanisms that regulate differentiation. This paper describes an improved approach to establish prostate organoids, and introduces methods to (1) collect protein lysate from organoids, and (2) fix and stain organoids for whole-mount confocal microscopy.

利用有机体培养评价小鼠前列腺皮细胞的分化能力

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation ...

Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary epithelium using transplantation techniques takes advantage of the hormonal environment provided by the adolescent rodent host. These studies are ideally suited to explore the impact of various biological manipulations on mammary gland development and dissect many aspects of mammary gland biology. A common, but limiting, feature is that transplanted epithelial cells are strongly influenced by the surrounding stroma and outcompeted by endogenous epithelium; to utilize native mammary tissue, the abdominal-inguinal white fat pad must be cleared to remove host mammary epithelium prior to the transplantation. A major obstacle when using the rat model organism is that clearing the developing mammary tree in post-weaned rats is not efficient. When transplanted into gland-free fat pads, donor epithelial cells can repopulate the cleared host fat pad and form a functional mammary gland. The interscapular fat pad is an alternative location for these grafts. A major advantage is that it lacks ductal structures yet provides the normal stroma that is necessary to promote epithelial outgrowth and is easily accessible in the rat. Another major advantage of this technique is that it is minimally invasive, because it eliminates the need to cauterize and remove the growing endogenous mammary tree. Additionally, the interscapular fat pad contains a medial blood vessel that can be used to separate sites for grafting. Because the endogenous glands remain intact, this technique can also be used for studies comparing the endogenous mammary gland to the transplanted gland. This paper describes the method of mammary epithelial cell transplantation into the interscapular white fat pad of rats.

This article describes a transplantation method to graft donor rat mammary epithelial cells into the interscapular white fat pad of recipient animals. This method can be used to examine host and/or donor effects on mammary epithelium development and eliminates the need for pre-clearing, thereby extending the usefulness of this technique.

大鼠乳腺内皮细胞移植到间膜白脂肪垫

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary ...