Name: characterization of cell membrane extensions and studying their roles in cancer cell adhesion dynamics
Uploaded: 2018-03-26T14:30:00
Description: Watch this Scientific Journal Video about Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics at JoVE.com

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1. 细胞扩展特性
<ol><li>在标准的37.0 摄氏度和 5% CO2中, 用10% 胎牛血清和 1万 U/毫升青霉素和链霉素 (指定为 "生长培养基"), 将 SW-13、SW 和 SW-DKK3 细胞维持在标准化的湿润孵化箱中, 并在 Dulbecco 的改良后的鹰培养基中补充。<ol>
<li>使用 hemocytometer 计数细胞, 并将5000个细胞 SW-13, SW-新, SW-DKK3 线分成单独的6井板, 在无菌玻璃盖玻片的生长培养基中一夜之间生长。</li>
	</ol>
</li>
	<li>在一夜之间生?...

<ol>
	<li>Libe, R., Fratticci, A., Bertherat, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adrenocortical+cancer:+pathophysiology+and+clinical+management.">Adrenocortical cancer: pathophysiology and clinical management.</a> Endocrine-related cancer. 14 (1), 13-28 (2007).</li><li>Veeck, J., Dahl, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+Wnt+pathway+in+cancer:+the+emerging+role+of+Dickkopf-3.">Targeting the Wnt pathway in cancer: the emerging role of Dickkopf-3.</a> Biochimica et biophysica acta. 1825 (1), 18-28 (2012).</li><li>Friedl, P., Gilmour, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+cell+migration+in+morphogenesis,+regeneration+and+cancer.">Collective cell migration in morphogenesis, regeneration and cancer.</a> Nat Rev Mol Cell Biol. 10 (7), 445-457 (2009).</li><li>Jacquemet, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-type+calcium+channels+regulate+filopodia+stability+and+cancer+cell+invasion+downstream+of+integrin+signalling.">L-type calcium channels regulate filopodia stability and cancer cell invasion downstream of integrin signalling.</a> Nat Commun. 7, 13297 (2016).</li><li>Machesky, L. M., Li, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fascin:+Invasive+filopodia+promoting+metastasis.">Fascin: Invasive filopodia promoting metastasis.</a> Commun Integr Biol. 3 (3), 263-270 (2010).</li><li>Petrie, R. J., Harlin, H. M., Korsak, L. I., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activating+the+nuclear+piston+mechanism+of+3D+migration+in+tumor+cells.">Activating the nuclear piston mechanism of 3D migration in tumor cells.</a> J Cell Biol. 216 (1), 93-100 (2017).</li><li>Korah, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+silencing+of+RASSF1A+deregulates+cytoskeleton+and+promotes+malignant+behavior+of+adrenocortical+carcinoma.">Epigenetic silencing of RASSF1A deregulates cytoskeleton and promotes malignant behavior of adrenocortical carcinoma.</a> Molecular cancer. 12, 87 (2013).</li><li>Cheng, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+FOXO1-mediated+dedifferentiation+blocking+role+for+DKK3+in+adrenocortical+carcinogenesis.">A novel FOXO1-mediated dedifferentiation blocking role for DKK3 in adrenocortical carcinogenesis.</a> BMC cancer. 17 (1), 164 (2017).</li><li>Barry, D. J., Durkin, C. H., Abella, J. V., Way, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+source+software+for+quantification+of+cell+migration,+protrusions,+and+fluorescence+intensities.">Open source software for quantification of cell migration, protrusions, and fluorescence intensities.</a> J Cell Biol. 209 (1), 163-180 (2015).</li><li>Cliffe, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+3D+analysis+of+complex+single+border+cell+behaviors+in+coordinated+collective+cell+migration.">Quantitative 3D analysis of complex single border cell behaviors in coordinated collective cell migration.</a> Nat Commun. 8, 14905 (2017).</li></ol>

在这里, 我们描述了一个体外定量的方法来表征细胞扩展的简便, 少坑瀑布, 可靠的重现性, 可用于各种测试条件。此外, 简单, 可量化的黏附力和/或运动分析可以同时执行, 以关联潜在的功能意义的观察改变的细胞膜延长。
然而, 这些方法可能会带来一些潜在的限制。首先, 这里指定的标准, 以确定不同类型的细胞扩展是基于肾上腺皮质 SW-13 细胞形态学, 因此可能需要细?...

失调 wnt 信号在肾上腺皮质恶性肿瘤中起重要作用1。本研究中使用的方法研究了 DKK3 的沉默, 即 WNT 信号的负调节因子, 是否代表肾上腺皮质的分化事件, 并在细胞扩展曲目变化的背景下促进肿瘤的形成。DKK3 是一个 38 kDa 分泌糖蛋白与 n-终端信号肽和以往的研究表明, 其强迫表达导致细胞周期骤停, 抑制侵袭性恶性行为, 并逆转上皮间充质转换2。
肾上腺皮质癌 (ACC) 和其他癌症的恶性行为在某种程度上受肿瘤细胞与周围表面相互作用的能力的影响, 包括细胞外基质, 进而促进肿瘤细胞侵袭和迁移3。特定细胞膜延伸在癌症进展中的作用正越来越多地表现在不同的语境中, 主要是通过丝状伪足的形成。例如, L 型钙通道的过度表达诱导丝状伪足形成, 促进肿瘤细胞侵袭 4.同样, 在正常组织中最小表达的肌动蛋白结合蛋白 Fascin 也抗原与丝状伪足形成5相关联的癌细胞.Lobopodia 的形成使非恶性成纤维细胞能够有效地通过超细胞基质迁移, 然而, 它已经表明, 肉瘤细胞依靠金属蛋白酶的活动代替 Lobopodia, 以促进细胞迁移和入侵6。我们已经表明, 肿瘤抑制器, 包括 Ras 协会域家族1异构体 A (RASSF1A) 和 DKK3, 可以发挥作用, 以改变骨架元素和促进 lamellipodia 形成和阻碍侵入性属性7,8。
因此, 重要的是要描述发生癌变的基因的影响及其与细胞膜延长改变的关系, 特别是评估丝状伪足、lobopodia 和 lamellipodia 在试验条件下的形成。目前的先进技术包括使用越来越复杂的显微方法、荧光标记和/或复杂的计算机算法来获取和解释数据。虽然这些方法提供了新的强大的分析工具, 但它们的复杂性限制了它们在细胞生物学实验中的广泛应用和适应性。此外, 对单元格扩展形态学变化的精确量化和观察通常不被测量9,10。相比之下, 我们在这里引入了一种技术, 通过标准显微技术和易于适应的体外方法, 精确地量化细胞扩展的变化。这些方法还量化每个细胞的扩展类型同时为每个细胞分析和确定的整体变化的细胞膜延长曲目。我们还展示了这些变化如何与细胞黏附性质有关。
作为一个实验性的例子, 我们将使用以前创建的 SW-13 细胞系, 指定的 SW-DDK3, 它已经稳定地转染了 pCMV6-Entry/DKK3 质粒载体和组成性 overexpresses DKK3, 一个区别因素在肾上腺。非转染的 SW-13 细胞和 SW-13 细胞稳定转染的空载体 (pCMV6-Entry), 指定的 SW, 将作为实验控制。

<table width="653">
	<tbody>
		<tr height="63">
			<td height="63" style="width:216px;height:63px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:115px;">ThermoFisher</td>
			<td style="width:119px;">11965-092</td>
			<td style="width:203px;">Supplemented with fetal bovine serum and penicllin and streptomycin</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">Deulbeco&#39;s Phosphate Buffered Saline</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td>1X solution, used directly</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">3.7% formaldehyde&#160;</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">F8775</td>
			<td>Dilute stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">0.05% crystal violet&#160;</td>
			<td style="width:115px;">Harleco</td>
			<td style="width:119px;">192-12</td>
			<td>Dilute stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">De-ionized water</td>
			<td style="width:115px;">NA</td>
			<td style="width:119px;">NA</td>
			<td>NA</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">Microscope with 400X magnification&#160;</td>
			<td style="width:115px;">NA</td>
			<td style="width:119px;">NA</td>
			<td>NA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-well plates</td>
			<td>Corning</td>
			<td>353046</td>
			<td>NA</td>
		</tr>
		<tr height="43">
			<td height="43" style="width:216px;height:43px;">Cell dissociation solution non-enzymatic 1X</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">C5914&#160;</td>
			<td>1X solution, used directly</td>
		</tr>
	</tbody>
</table>

characterization of cell membrane extensions and studying their roles in cancer cell adhesion dynamics

细胞膜的扩展曲目调节癌细胞的各种恶性行为, 包括它们的粘合剂和迁移电位。准确分类和量化细胞扩展的能力和测量细胞的粘附能力对确定细胞信号事件如何影响癌细胞的行为和侵略性至关重要。在这里, 我们描述了体外的设计和使用的细胞扩展量化方法结合的黏附能力检测在建立的体外模型的肾上腺皮质癌 (ACC)。具体来说, 我们测试 DKK3, 一个假定的肿瘤抑制物和赞成分化因子, 对 ACC 细胞系, SW-13 的膜扩张表型的影响。我们建议这些化验提供相对简单, 可靠, 容易解释的指标, 以衡量这些特点在各种实验条件下。

采用上述检测方法, 对建立的 ACC 细胞系 SW-13、体外进行了 DKK3 过度表达对细胞增殖形态学和细胞黏附性能的影响。细胞 overexpressing DKK3 是由稳定的转染 SW-13 细胞与 c-myc DDK 标记 pCMV6-Entry/DKK3 质粒载体, 并指定为 SW-DKK3。同样地, 以空矢量 pCMV6-Entry 稳定转染的细胞也被创建为传代控制, 并被定义为 SW-尼奥。SW-13 细胞被用作额外的父细胞控制。定量实时 PCR 和西方印迹技...

Watch this Scientific Journal Video about Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics at JoVE.com

Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics

本研究表明定量细胞膜延伸测量的实用性和易用性, 及其与细胞粘附能力的相关性。作为一个代表性的例子, 我们在这里表明, Dickkopf 相关蛋白 3 (DKK3) 促进增加 lobopodia 形成和细胞粘附在肾上腺皮质癌细胞在体外。

细胞膜延伸特性及其在癌细胞黏附动力学中的作用研究

The cell membrane&#39;s extension repertoire modulates various malignant behaviors of cancer cells, including their adhesive and migratory potentials. The ...

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