Ohse 赠款基金会资助了这项工作。

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>在一夜之间生长, 为每井, 吸入生长培养基, 洗涤细胞与1毫升温暖磷酸盐缓冲盐水 (PBS), 然后修复细胞与1毫升3.7% 甲醛为10分钟。</li>
	<li>吸入甲醛和染色细胞在每个井与1毫升0.05% 水晶紫30分钟. 用三洗涤1毫升去离子水清洗过量染料。 注意: 根据染料摄取的水平, 可能需要额外的一些洗涤来去除背景染色, 以促进细胞的可视化。<ol>
<li>使用细尖钳, 检索盖玻片, 并把他们脸上的标签玻璃幻灯片与一滴清晰的安装试剂。</li>
	</ol>
</li>
	<li>在光镜下, 随机选择20个不同的细胞, 从每个盖玻片的细胞延伸试验。<ol>
<li>以每个视野的400x 放大倍数显微照片。直接计算20个代表单元格中每个扩展类型的数目。 注: 因此, 应检查每一个测试的条件, 20 个独立的细胞, 以确保可靠的结果。丝状伪足是由细胞扩展定义的, 其基部为5微米或更小, 单顶点。Lobopodia 是由细胞扩展定义的, 其基数为5到20微米, 包含有多个顶端的扩展。Lamellipodia 的定义是细胞扩展, 长度大于20微米, 有或没有顶端。根据已测试的单元格类型, 单元格扩展的大小可能会有所不同, 并且可能需要对识别参数进行细化。</li>
	</ol>
</li>
	<li>确定每个单元格类型中每个单元格扩展的平均数目 (即、SW-13、sw-DDK), 并检查了6个水井。</li>
	<li>采用单向方差分析 (方差) 统计试验, 比较不同试验细胞类型间细胞膜延长平均百分率的差异。</li>
</ol>2. 黏附力试验
<ol><li>在37.0 摄氏度和 5% CO2的生长培养基中, 维持 SW-13、SW 和 SW-DKK3 细胞。<ol>
<li>使用 hemocytometer, 计数和板10万细胞的 SW-13, SW-新, 和 SW-DKK3 每井成单独的6井板和生长在一夜之间生长培养基。</li>
		<li>种子6井板为每个细胞类型测试, 使用一个板材为每6指定的时间点测试下面。 注意: 不同的单元格类型的时间点可能会有所不同。</li>
	</ol>
</li>
	<li>夜间孵化后, 用温暖的 PBS 冲洗细胞, 然后加入0.5 毫升的1x 非酶细胞离解溶液, 每一个井。<ol>
<li>旋转板块, 传播细胞离解溶液。在已定义的时间点 (即1、2、3、5、10和15分钟) 中吸入指定的板, 然后用1毫升温 PBS 解决方案三次清洗。</li>
		<li>在洗涤之间, 轻轻地敲击盘子以分离松散附着的细胞。</li>
	</ol>
</li>
	<li>吸入任何剩余的 PBS, 并固定在板上剩余的细胞与1毫升3.7% 甲醛10分钟。</li>
	<li>染色细胞与1毫升0.05% 晶体紫30分钟然后用1毫升的去离子水洗涤多余的染料。滴定洗涤促进细胞可视化。 注意: 根据染料摄取的水平, 可能需要额外的一些洗涤来促进细胞的可视化。</li>
	<li>使用光显微镜在100x 放大倍数, 计数剩余的连接的细胞在每个井为每个板材。 注意: 使用透明标尺或在板的底部标记细笔参考线将有助于避免重复计数相同的单元格。</li>
	<li>确定每个板块每个井的平均附着单元数。</li>
	<li>采用双向方差分析统计试验, 比较 SW-13、SW、SW-DDK3 细胞在给定时间段内的细胞脱离率。</li>
</ol>

<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>

作者没有什么可透露的。

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 ...

characterization-cell-membrane-extensions-studying-their-roles-cancer

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Cancer Research

7.4K 次观看.  Yale University School of Medicine. 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。

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

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a viable treatment strategy and biopsy is not routinely performed, the availability of patient samples for research is limited. Consequently, efforts to study this disease have been challenged by a paucity of faithful disease models. To address this need, we describe here a protocol for the rapid processing of post-mortem autopsy tissue samples in order to generate durable patient-derived cell culture models that can be used in in vitro assays or in vivo orthotopic xenograft experiments. These models can be used to screen for potential drug targets and to study fundamental pathobiological processes within DIPG. This protocol can further be extended to analyze and isolate tumor and microenvironmental cells using Fluorescence-activated Cell Sorting (FACS), which enables subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA at the bulk cell or single cell level. Finally, this protocol can also be adapted to generate patient-derived cultures for other central nervous system tumors.

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma DIPG

This protocol describes a method for the rapid processing of post-mortem diffuse intrinsic pontine glioma samples for the establishment of patient-derived cell culture models or direct characterization of tumor and microenvironmental cells.

一种内在的弥漫性脑胶质瘤脑桥快速验尸细胞培养协议（DIPG）

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a ...

科研

癌症研究

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of nuclear apoptotic characteristics, including chromatin condensation and DNA breakage. Once the MMP collapses, cell apoptosis will initiate irreversibly. A series of lipophilic cationic dyes can pass through the cell membrane and aggregate inside the matrix of mitochondrion, and serve as fluorescence marker to evaluate MMP change. As one of the six members of the Cl- intracellular channel (CLIC) family, CLIC4 participates in the cell apoptotic process mainly through the mitochondrial pathway. Here we describe a detailed protocol to measure MMP via monitoring the fluorescence fluctuation of Rhodamine 123 (Rh123), through which we study apoptosis induced by CLIC4 knockdown. We discuss the advantages and limitations of the application of confocal laser scanning and normal fluorescence microscope in detail, and also compare it with other methods.

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Here we present a detailed protocol for the application of rhodamine 123 to identify the mitochondrial membrane potential (MMP) and study CLIC4 knockdown-induced HN4 cell apoptosis in vitro. Under common fluorescence microscope and confocal laser scanning fluorescence microscope, the real-time change of the MMP was recorded.

线粒体膜电位检测 CLIC4 击倒诱导 HN4 细胞凋亡的实验研究

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of ...

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

单细胞下游特征的靶细胞预富集与全基因组扩增

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. While 75% of bladder cancers are non-invasive and unlikely to metastasize, about 25% progress to an invasive growth pattern. Up to half of the patients with invasive cancers will develop lethal metastatic relapse. Thus, understanding the mechanism of invasive progression in bladder cancer is crucial to predict patient outcomes and prevent lethal metastases. In this article, we present a three-dimensional cancer invasion model which allows incorporation of tumor cells and stromal components to mimic in vivo conditions occurring in the bladder tumor microenvironment. This model provides the opportunity to observe the invasive process in real time using time-lapse imaging, interrogate the molecular pathways involved using confocal immunofluorescent imaging and screen compounds with the potential to block invasion. While this protocol focuses on bladder cancer, it is likely that similar methods could be used to examine invasion and motility in other tumor types as well.

The processes governing bladder cancer invasion represent opportunities for biomarker and therapeutic development. Here we present a bladder cancer invasion model which incorporates 3-D culture of tumor spheroids, time-lapse imaging and confocal microscopy. This technique is useful for defining the features of the invasive process and for screening therapeutic agents.

3维细胞培养系统研究膀胱癌侵袭和评价治疗方法

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

骨髓间充质干细胞在浆组织中的分离及与癌细胞联合培养研究其相互作用

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

建立胃癌患者衍生异种移植模型和原细胞系

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

免疫荧光对非小细胞肺癌患者循环肿瘤细胞的半自动PD-L1特征化及枚举

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

Radiosensitivity of Cancer Stem Cells in Lung Cancer Cell Lines

The presence of cancer stem cells (CSCs) has been associated with relapse or poor outcomes after radiotherapy. Studying radioresistant CSCs may provide clues to overcoming radioresistance. Voltage-gated calcium channel &#945;2&#948;1 subunit isoform 5 has been reported as a marker for radioresistant CSCs in non-small cell lung cancer (NSCLC) cell lines. Using calcium channel &#945;2&#948;1 subunit as an example of a CSC marker, methods to study the radiosensitivity of CSCs in NSCLC cell lines are presented. CSCs are sorted with putative markers by flow cytometry, and the self-renewal capacity of sorted cells is evaluated by sphere formation assay. Colony formation assay, which determines how many cells lose the ability to generate descendants forming the colony after a certain dose of radiation, is then performed to assess the radiosensitivity of sorted cells. This manuscript provides initial steps for studying the radiosensitivity of CSCs, which establishes the basis for further understanding of the underlying mechanisms.

The presence of cancer stem cells have been associated with relapse or poor outcomes after radiotherapy. This manuscript describes the methods to study the radiosensitivity of cancer stem cells in lung cancer cell lines.

肺癌细胞系中癌症干细胞的放射敏感性

The presence of cancer stem cells (CSCs) has been associated with relapse or poor outcomes after radiotherapy. Studying radioresistant CSCs may provide ...

Comparing Metastatic Clear Cell Renal Cell Carcinoma Model Established in Mouse Kidney and on Chicken Chorioallantoic Membrane

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, one third of ccRCC patients will develop metastases to the lung, which is related to a very poor outcome for patients. Unfortunately, no therapy is available for this deadly stage, because the molecular mechanism of metastasis remains unknown. It has been known for 25 years that the loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene is pathognomonic of ccRCC. However, no clinically relevant transgenic mouse model of ccRCC has been generated. The purpose of this protocol is to introduce and compare two newly established animal models for metastatic ccRCC. The first is renal implantation in the mouse model. In our laboratory, the CRISPR gene editing system was utilized to knock out the VHL gene in several RCC cell lines. Orthotopic implantation of heterogeneous ccRCC populations to the renal capsule created novel ccRCC models that develop robust lung metastases in immunocompetent mice. The second model is the chicken chorioallantoic membrane (CAM) system. In comparison to the mouse model, this model is more time, labor, and cost-efficient. This model also supported robust tumor formation and intravasation. Due to the short 10 day period of tumor growth in CAM, no overt metastasis was observed by immunohistochemistry (IHC) in the collected embryo tissues. However, when tumor growth was extended by two weeks in the hatched chicken, micrometastatic ccRCC lesions were observed by IHC in the lungs. These two novel preclinical models will be useful to further study the molecular mechanism behind metastasis, as well as to establish new, patient-derived xenografts (PDXs) toward the development of novel treatments for metastatic ccRCC.

Metastatic clear cell renal cell carcinoma is a disease without a comprehensive animal model for thorough preclinical investigation. This protocol illustrates two novel animal models for the disease: the orthotopically implanted mouse model and the chicken chorioallantoic membrane model, both of which demonstrate lung metastasis resembling clinical cases.

比较在小鼠肾脏和鸡胆膜上建立的转移性透明细胞肾细胞癌模型

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, ...

Quantification of Tumor Cell Adhesion in Lymph Node Cryosections

Tumor-draining lymph nodes (LNs) are not merely filters of tumor-produced waste. They are one of the most common regional sites of provisional residence of disseminated tumor cells in patients with different types of cancer. The detection of these LN-residing tumor cells is an important biomarker associated with poor prognosis and adjuvant therapy decisions. Recent mouse models have indicated that LN-residing tumor cells could be a substantial source of malignant cells for distant metastases. The ability to quantify the adhesivity of tumor cells to LN parenchyma is a critical gauge in experimental research that focuses on the identification of genes or signaling pathways relevant for lymphatic/metastatic dissemination. Because LNs are complex 3D structures with a variety of appearances and compositions in tissue sections depending on the plane of section, their matrices are difficult to replicate experimentally in vitro in a fully controlled way. Here, we describe a simple and inexpensive method that allows the quantification of adhesive tumor cells to LN cryosections. Using serial sections of the same LN, we adapt the classic method developed by Brodt to use nonradioactive labels and directly count the number of adhering tumor cells per LN surface area. LN-adherent tumor cells are readily identified by light microscopy and confirmed by a fluorescence-based method, giving an adhesion index that reveals the cell-binding affinity to LN parenchyma, which is suggestive evidence of molecular alterations in the affinity binding of integrins to their correlate LN-ligands.

Here, we describe a simple and inexpensive method that allows the quantification of adhesive tumor cells to lymph node (LN) cryosections. LN-adherent tumor cells are readily identified by light microscopy and confirmed by a fluorescence-based method, giving an adhesion index that reveals the tumor cell-binding affinity to LN parenchyma.

淋巴结冷冻科肿瘤细胞粘附的定量

Tumor-draining lymph nodes (LNs) are not merely filters of tumor-produced waste. They are one of the most common regional sites of provisional residence ...