这项研究得到了格兰特号的支持。CA-81301 来自国家癌症研究所我们感谢鲍勃的编辑协助, Dr. 彼得 Villalta 和鲁迅的质谱协助分析生物化学共享资源共济会癌症中心, 和 Dr. 亚当 t. Zarth 和 Dr. 安娜 k. 米歇尔的宝贵讨论和输入。分析生物化学共享资源由国家癌症研究院癌症中心资助 CA-77598 部分支持

1. 材料和常规过程 <ol> <li> 将 NNN 合成为以前描述的 15 。获得 norcotinine, P450 2A6 Baculosomes, 再生系统, 0.5x 和 #160; 反应缓冲器, 以及试剂级商业来源的所有其他化学品或溶剂. </li>
	<li> 在 500 MHz 光谱仪上记录核磁共振谱。报告化学变化为每百万分之 (ppm)。使用残余溶剂峰值作为内部参考为 1 H-nmr (7.26 ppm CDCl 3 ) 和 13 C-nmr (77.2 ppm CDCl 3 ). 
	注意: 峰值分裂使用了以下简称: s = 单线态, d = 双峰, dd = 双峰的双峰, dt = 三胞胎的双峰, dq = 双峰的四重奏, ddd = 双峰的双峰, 和 m = multiplet. </li>
	<li> 对 LTQ Orbitrap Velos 上所选化合物执行高分辨率质谱 (人力资源), 并将数据报告为 m/z . </li>
	<li> 使用金预硅胶薄层色谱片 (40 毫米 x 80 毫米, 0.2 毫米厚) 与 254 nm 荧光指示剂 (tlc)。通过 UV 灯照射来可视化薄层色板. </li>
	<li> 在60和 #197 上执行闪光色谱; 70-150 目硅胶使用 1.5 cm x 15 cm 玻璃柱. </li>
</ol> 2。nitrosamide 阳性控制 ( N & #8242; -nitrosonorcotinine, NNC) <ol> <li> 干燥, 在烤箱过夜 (16 小时, 〜140和 #176; C), 一个25毫升, 圆底烧瓶含有磁性搅拌棒。早上, 在 n 2 流下冷却烧瓶, 同时使用油起泡使 n 2 流速保持在每秒一个气泡下. 
	注: 冷却后, 不需要在 N 2 下保持烧瓶的预防措施. </li>
	<li> 在通风罩中添加 norcotinine (31.8 毫克、0.196 摩尔)、醋酸酐 (5 毫升) 和乙酸 (1 毫升) 到冷却烧瓶中。在冷却到0和 #176 时, 将此混合物连续搅拌; C 冰浴. 
	 警告: 醋酸酐和乙酸具有腐蚀性. </li>
	<li> 添加纳米 2 (33.3 毫克, 0.483 摩尔) 在一个部分和连续搅拌的混合物为2.5 小时. 监测反应进展的 TLC (100% EtOAc, R f = 0.19, 参见步骤 1.4). 
	注: 在这段时间, 混合物将变得越来越黄与偶尔起泡. </li>
	<li> 通过将混合物倒入 ice-cold H 2 O (18 毫升) 来淬火反应。在 100 ml 分漏斗中, 立即用18毫升 CH 2 Cl 2 提取水溶液。用 CH 2 Cl 2 (9 毫升) 将水层至少再抽出2次. </li>
	<li> 干燥100毫克 MgSO 的混合有机物 4 用于2分钟的过滤, 并通过旋转蒸发浓缩溶液以产生原油、黄油。在蒸发过程中, 将水浴加热到30和 #176; C. 
	注意: 如果化合物在 CH 2 Cl 2 中被溶解, 并且该解决方案在黑暗中存储在 2-8 和 #176 中, 则可以暂停该协议; C. </li>
	<li> 用硅胶作为固定相和 100% EtOAc 为淋洗 16 , 通过柱层析 (见步骤 1.5) 净化粗化合物. 
	 警告: NNC 和相关的 nitrosamides 应小心处理, 因为它们被认为是人类致癌物. </li>
	<li> 溶解 CDCl 中的纯化合物 3 并使用 NMR (参见步骤 1.2) 确认结构并确定此解决方案的浓度 17 。将此解决方案存储在 2-8 和 #176 的黑暗中, 直到需要为止. 
	注意: 此解决方案将用作 体外 检测 (步骤 3.5) 中的正向控制。在支持信息中提供了核磁共振波谱和化学位移. </li>
	<li> 使用纯 NNC 解决方案, 通过在步骤1.3 和4.3 中描述的参数直接注入高分辨率质谱仪 (人力资源), 确定准确的母体质量并测定产品离子质量. 
	注: 产品质量将用于检测该化合物在 体外 代谢试验 (步骤 4.3). </li>
</ol> 3。一个例子 nitrosamine-P450 体外 孵化 <ol> <li> 删除 P450 2A6 Baculosomes, 生动-再生系统, 和 10 mM NADP + 库存解决方案从-80 和 #176; C 冷冻, 让他们解冻冰。一旦解冻, 稀释 Baculosomes 和再生系统1:10 和1:50 分别, 成一个单一的1毫升管与0.5X 反应缓冲器。同样, 添加3和 #956; NADP + 库存解决方案为97和 #956; l 0.5X 反应缓冲器. 
	注意: NADP + 是光敏的。通过将容器包装在铝箔中来保护此解决方案。酶解对冻融循环敏感;将此限制为不超过2个周期。为将来的实验做必要的准备等分. </li>
	<li> 对于每个孵化, 添加50和 #956; 酶溶液 l (含 5 pmol P450) 至1毫升管, 包含40和 #956; l 4 和 #956; M NNN 的解决方案, 由反应缓冲器。预孵化这种新的混合物为2分钟, 在37和 #176; C 使用水浴, 然后添加10和 #956; 稀释的 NADP + 解决方案。孵育整个系统 1-30 分钟, 在37和 #176; C. 
	注: 使用5分钟的潜伏期 (如 5, 10, 15 分钟等) 将提供足够的 nitrosamide 形成时间的过程. </li>
	<li> 在所需的孵育时间后, 先将10和 #956 进行淬火; 3.0 n ZnSO 4 , 然后是10和 #956; 3.0 n Ba (哦) 2 的 l. 漩涡这个解决方案和白色沉淀将形成。离心样品在 8000 x g 为4分钟颗粒的沉淀. 
	注: 此步骤不能暂停, 因为形成的 nitrosamides 在水溶液中的稳定性有限。长时间的停顿可能导致错误的底片. </li>
	<li> 立即将上清液移出, 并分析2和 #956; 用液体色谱法正 nanoelectrospray 电离高分辨串联质谱法 (LC-NSI + -人力资源/毫秒, 步骤 4.1-4.3). </li>
	<li> 对于正控制孵化, 将含有 100 fmol 的合成 NNC 溶液的分蒸发到1毫升的管下, 在 N 的流下为 2 。对这个小瓶, 添加完整的酶系统从步骤3.2 和继续按照描述的步骤 3.3-3.4. </li>
	<li> 对于负控制孵化, 执行所描述的实验 (步骤 3.2-3.4), 除替换50和 #956; L 酶溶液与0.5X 反应缓冲器. </li>
</ol> 4。用于 lc 组件的 lc-NSI + -人力资源/MS <ol> <li> 的 nitrosamide 检测参数的示例, 请将步骤4.2 中的以下多级渐变应用于使用商用、手动包装的 18 C18 (5 和 #956; m) 的 UPLC 系统,100毫米 x 75 和 #956; m, 15 和 #956; m 孔毛细管柱. </li>
	<li> 使用 5 mM NH 4 华侨作为溶剂 a 和 MeCN 作为溶剂 b, 通过在1和 #956 的 5% b 处运行, 将样品加载到列上; L/分钟从 0-5 分钟, 然后减慢流速到0.3 和 #956; 之后的升/分钟。接下来, 运行从5到 20% b 以上4分钟的渐变, 后跟一个斜坡到 55% b 以上10分钟, 然后 re-equilibration 到 5% b. </li>
	<li> 在 LTQ Orbitrap 上执行 LC-NSI + -人力资源/MS。通过全扫描和 MS 2 碎片监视 NNC。以6万的分辨率进行全扫描, 并以 5 ppm 的质量公差提取准确的母质量 (192.07670)。使用 ms 2 碎片分离父离子 (2.0) 和由碰撞引发的离解 (CID) 与 25 eV 的碰撞能量, 分辨率 1.5万, 和扫描时间30毫秒提取的产品离子的准确质量 NNC ( m/z 192 m/z 134.04739 和 162.07874) 的质量公差为 5 ppm. </li>
</ol>

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作者没有什么可透露的。

阐明亚硝胺的代谢是了解其致癌性的重要组成部分。由于涉及的细胞色素 P450s 和其他代谢酶是多态的, 进一步应用这一知识可能会发现高危个体1,4。新的数据表明, 进一步氧化α-hydroxynitrosamines, 推定主要代谢物的亚硝胺参与 DNA 结合, 以 nitrosamides 是可能的;然而, 这并没有在广泛的基板上进行强有力的测试。我们已经描述了一个协议来确定性氧化亚硝胺的...

N-亚硝胺是一种在饮食、烟草制品和一般环境中发现的大量致癌物质;它们也可以在人体内形成内1。超过 300 N-硝基化合物已经被测试和 #62; 90% 被评估了作为致癌物在动物模型2,3。为了展示它们的致癌性, 这些化合物必须首先激活细胞色素 P450s1,2,3。研究表明, 细胞色素 P450s 容易氧化亚硝胺为α-hydroxynitrosamines (图 1), 这是高度活性化合物与半衰期的〜 5 s 之前自发分解到 alkyldiazohydroxides。后者可以在丢失 H2O 和 N2之后烷基化 DNA。由此产生的 DNA 加, 如果未, 可以导致突变, 如果在关键的 onco 或抑癌基因, 导致癌症的发展1。因此, 为了充分了解细胞色素 P450 氧化致癌亚硝胺的代谢途径、DNA 加和下游代谢产物, 已经花费了大量的精力。这些知识在个人癌症风险评估中具有潜在的应用价值4。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56312/56312fig1.jpg"> 
图 1: 一般和建议的亚硝胺代谢。 亚硝胺 (1) 被 P450s 氧化为α-hydroxynitrosamines (2), 自发分解为 alkyldiazohydroxides (3)。这些化合物可以与 dna 结合形成 dna 加。假设2被 P450s 进一步氧化为 nitrosamides 4。这些可以直接绑定到 dna, 形成新的 dna 加或水解到3 , 形成已知的 dna 加。r1和 r2表示任何烷基组。<a href="//ecsource.jove.com/files/ftp_upload/56312/56312fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
虽然α-hydroxynitrosamine 假说得到了大量数据的坚实支持, 但也存在一些不一致之处。主要的一个是α-hydroxynitrosamines 的短半衰期5,6。众所周知, 这些化合物是在内质网膜和后来的烷基化核 DNA 中产生的。鉴于他们的生命的几秒钟, 这是令人费解的这些中间体如何生存所需的旅行, 虽然胞。一个假说是, α-hydroxynitrosamines 的一部分被 processively 氧化为 nitrosamides7,8, 在比较9中相当稳定。这可能会发生通过保留α-hydroxynitrosamines 在细胞色素 P450 活性部位。这种类型的氧化的先例已经看到与尼古丁10, 酒精的11, 和简单的 alkylnitrosamines12,13。另外, nitrosamides 是直接作用的致癌物质2,3。根据它们的反应性9, 这些化合物被认为产生的 dna 加与α-hydroxynitrosamines 和新的, 未经探索的 dna 加 (图 1) 的结果相同。因此, 这一假说不仅解释了通过胞的运输, 也说明了 DNA 损伤产物的形成。
本文介绍了一种用于评价亚硝胺的细胞色素 P450-mediated 转化为 nitrosamides 的体外的通用协议。先前报告的′-nitrosonornicotine (NNN) 转换为′-nitrosonorcotinine (NNC) 的细胞色素 P450 2A6 被展示为示例14。该协议在广泛的基质酶系统的应用将有助于确定 nitrosamides 在整体硝代谢中的重要性。

<table>
	<tbody>
		<tr>
			<td>Norcotinine</td>
			<td>AKoS GmbH (Steinen, Germany)</td>
			<td>CAS 17708-87-1, AKoS AK0S006278969</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>695092</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Anhydride</td>
			<td>Sigma-Aldrich</td>
			<td>242845</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>431311</td>
			<td></td>
		</tr>
		<tr>
			<td>Barium Hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>433373</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>151823</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC Acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td>34998</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylene Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>34856</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Nitrite</td>
			<td>Sigma-Aldrich</td>
			<td>237213</td>
			<td></td>
		</tr>
		<tr>
			<td>ViVid CYP2A6 Blue Screening Kit</td>
			<td>Life Technologies</td>
			<td>PV6140</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>221376</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 mL tubes</td>
			<td>Fisher</td>
			<td>AB0533</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mL round bottom flask</td>
			<td>Sigma-Aldrich</td>
			<td>Z510424</td>
			<td></td>
		</tr>
		<tr>
			<td>125 mL Erlenmeyer flask</td>
			<td>Sigma-Aldrich</td>
			<td>CLS4980125</td>
			<td></td>
		</tr>
		<tr>
			<td>125 mL Separatory Funnel</td>
			<td>Sigma-Aldrich</td>
			<td>Z261017</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL round bottom flask</td>
			<td>Sigma-Aldrich</td>
			<td>Z278262</td>
			<td></td>
		</tr>
		<tr>
			<td>500 MHz NMR Spectrometer</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Allegra X-22R Centrifuge</td>
			<td>Beckman-Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LC vials</td>
			<td>ChromTech</td>
			<td>CTC&#8211;0957&#8211;BOND</td>
			<td></td>
		</tr>
		<tr>
			<td>LTQ Orbitrap Velos</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Stir bar</td>
			<td>Sigma-Aldrich</td>
			<td>Z127035</td>
			<td></td>
		</tr>
		<tr>
			<td>NMR tube</td>
			<td>Sigma-Aldrich</td>
			<td>Z274682</td>
			<td></td>
		</tr>
		<tr>
			<td>P1000, P200, and P10 pipettes</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary evaporator</td>
			<td>Sigma-Aldrich</td>
			<td>Z691410</td>
			<td></td>
		</tr>
		<tr>
			<td>RSLCnano UPLC system</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Shaking Water Bath</td>
			<td>Fisher</td>
			<td>FSSWB15</td>
			<td></td>
		</tr>
		<tr>
			<td>Stir plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS6795420</td>
			<td></td>
		</tr>
		<tr>
			<td>PicoFrit Column</td>
			<td>New Objective</td>
			<td>PF3607515N5</td>
			<td></td>
		</tr>
		<tr>
			<td>Luna C18, 5 um</td>
			<td>Phenomenex</td>
			<td>535913-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

a general method for detecting nitrosamide formation in the in vitro metabolism of nitrosamines by cytochrome p450s

N-亚硝胺是一种公认的环境致癌物质, 需要细胞色素 P450 氧化来表现活性。代谢活化机制包括自发分解为 DNA 烷剂的α-hydroxynitrosamines 的形成。DNA 损伤的积累和由此产生的突变最终会导致癌症。新的证据表明, α-hydroxynitrosamines 可以进一步氧化到 nitrosamides processively 细胞色素 P450s。因为 nitrosamides 一般比α-hydroxynitrosamines 更稳定, 也可以烷基化 DNA, nitrosamides 可能在癌变中起到作用。在本报告中, 我们描述了一个一般的协议, 评估 nitrosamide 生产从体外细胞色素 P450-catalyzed 代谢亚硝胺。该协议采用了一般的方法合成相关的 nitrosamides 和一个体外细胞色素 P450 代谢测定的液相色谱-nanospray 电离-高分辨率串联质谱检测。此方法在示例研究中检测到′-nitrosonorcotinine 作为′nitrosonornicotine 的次要代谢物。该方法具有较高的灵敏度和选择性, 是由于质量检测准确。该方法在多种硝细胞色素 P450 系统中的应用将有助于确定这种转化的普遍性。由于细胞色素 P450s 的多态性和活动性的变化, 更好地了解 nitrosamide 的形成有助于个别癌症风险评估。

基于白色et al的工作。19, norcotinine 是硝化 NNC 清洁和高产 (80-92%), 以产生一个标准的体外实验。一个成功的反应的结构证据获得了从光谱分析包括1H 核磁共振, 13C 核磁共振, 舒适, 和 HSQC (支持信息) 与人力资源证实了父质量 [M + h]+在 5 ppm 的理论值 (图 2)。MS2的碎片 NNC 被用来确定最丰富的?...

Watch this Scientific Journal Video about A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s at JoVE.com

A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

细胞色素 P450s 致癌亚硝胺的α-羟基化是公认的代谢途径, 产生 DNA 损伤的中间体, 导致突变。然而, 新的数据表明进一步的氧化 nitrosamides 可能发生。我们描述了一个一般的方法, 检测 nitrosamides 产生的体外细胞色素 P450-catalyzed 代谢的亚硝胺。

用细胞色素 P450s 检测亚硝胺的体外Nitrosamide 形成的一般方法

N-nitrosamines are a well-established group of environmental carcinogens, which require cytochrome P450 oxidation to exhibit activity. The accepted ...

a-general-method-for-detecting-nitrosamide-formation-vitro-metabolism

Research

JoVE Journal

Cancer Research

A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

9.9K 次观看.  University of Minnesota. 细胞色素 P450s 致癌亚硝胺的α-羟基化是公认的代谢途径, 产生 DNA 损伤的中间体, 导致突变。然而, 新的数据表明进一步的氧化 nitrosamides 可能发生。我们描述了一个一般的方法, 检测 nitrosamides 产生的体外细胞色素 P450-catalyzed 代谢的亚硝胺。

视频: A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

该<em&gt;果蝇</em&gt;成虫盘瘤模型:基因的表达与肿瘤侵袭性的可视化和定量遗传马赛克

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

科研

癌症研究

Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates

Tumor models that closely imitate in vivo conditions are becoming increasingly popular in drug discovery and development for the screening of potential anti-cancer drugs. Multicellular tumor spheroids (MCTSes) effectively mimic the physiological conditions of solid tumors, making them excellent in vitro models for lead optimization and target validation. Out of the various techniques available for MCTS culture, the liquid-overlay method on agarose is one of the most inexpensive methods for MCTS generation. However, the reliable transfer of MCTS cultures using liquid-overlay for high-throughput screening may be compromised by a number of limitations, including the coating of microtiter plates (MPs) with agarose and the irreproducibility of uniform MCTS formation across wells. MPs are significantly prone to edge effects that result from excessive evaporation of medium from the exterior of the plate, preventing the use of the entire plate for drug tests. This manuscript provides detailed technical improvements to the liquid-overlay technique to increase the scalability and reproducibility of uniform MCTS formation. Additionally, details on a simple, semi-automatic, and universally applicable software tool for the evaluation of MCTS features after drug treatment is presented.

The uneven loss of medium from microtiter plates affects the reproducibility of uniform multicellular tumor spheroid formation. Improving culture conditions to reduce significant medium loss will improve the reproducibility of spheroid formation and the results of spheroid-based assays using the liquid-overlay technique.

蒸发减少培养条件的提高多细胞球体形成的微孔板重现

Tumor models that closely imitate in vivo conditions are becoming increasingly popular in drug discovery and development for the screening of potential ...

A Unified Methodological Framework for Vestibular Schwannoma Research

Vestibular schwannomas are the most common neoplasms of the cerebellopontine angle, making up 6-8% percent of all intracranial growths. Though these tumors cause sensorineural hearing loss in up to 95% of affected individuals, the molecular mechanisms underlying this hearing loss remain elusive. This article outlines the steps established in our laboratory to facilitate the collection and processing of various primary human tissue samples for downstream research applications integral to the study of vestibular schwannomas. Specifically, this work describes a unified methodological framework for the collection, processing, and culture of Schwann and schwannoma cells from surgical samples. This is integrated with parallel processing steps now considered essential for current research: the collection of tumor and nerve secretions, the preservation of RNA and the extraction of protein from collected tissues, the fixation of tissue for the preparation of sections, and the exposure of primary human cells to adeno-associated viruses for application to gene therapy. Additionally, this work highlights the translabyrinthine surgical approach to collect this tumor as a unique opportunity to obtain human sensory epithelium from the inner ear and perilymph. Tips to improve experimental quality are provided and common pitfalls highlighted.

The goal of this protocol is to outline the collection and processing of human surgical samples for multiple downstream applications in vestibular schwannoma and Schwann cell research.

前庭神经鞘瘤研究的统一方法框架

Vestibular schwannomas are the most common neoplasms of the cerebellopontine angle, making up 6-8% percent of all intracranial growths. Though these ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

向上移动: 一个简单而灵活的体外三维入侵检测协议

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction pathways and is tightly regulated by kinases and phosphatases. Therefore, characterizing the phosphoproteome may provide insights into identifying novel targets and biomarkers for oncologic therapy. Mass spectrometry provides a way to globally detect and quantify thousands of unique phosphorylation events. However, phosphopeptides are much less abundant than non-phosphopeptides, making biochemical analysis more challenging. To overcome this limitation, methods to enrich phosphopeptides prior to the mass spectrometry analysis are required. We describe a procedure to extract and digest proteins from tissue to yield peptides, followed by an enrichment for phosphotyrosine (pY) and phosphoserine/threonine (pST) peptides using an antibody-based and/or titanium dioxide (TiO2)-based enrichment method. After the sample preparation and mass spectrometry, we subsequently identify and quantify phosphopeptides using liquid chromatography-mass spectrometry and analysis software.

This protocol describes a procedure to extract and enrich phosphopeptides from prostate cancer cell lines or tissues for an analysis of the phosphoproteome via mass spectrometry-based proteomics.

磷酸浓缩结合无标签定量质谱法研究前列腺癌的 Phosphoproteome

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction ...

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic alterations in oncogenes such as the KRAS oncogene, which constitutes ~25% of lung cancer cases. The difficulty in therapeutically targeting KRAS-driven lung cancer partly stems from having poor models that can mimic the progression of the disease in the lab. We describe a method that permits the relative quantification of primary KRAS lung tumors in a Cre-inducible LSL-KRAS G12D mouse model via ultrasound imaging. This method relies on brightness (B)-mode acquisition of the lung parenchyma. Tumors that are initially formed in this model are visualized as B-lines and can be quantified by counting the number of B-lines present in the acquired images. These would represent the relative tumor number formed on the surface of the mouse lung. As the formed tumors develop with time, they are perceived as deep clefts within the lung parenchyma. Since the circumference of the formed tumor is well-defined, calculating the relative tumor volume is achieved by measuring the length and width of the tumor and applying them in the formula used for tumor caliper measurements. Ultrasound imaging is a non-invasive, fast and user-friendly technique that is often used for tumor quantifications in mice. Although artifacts may appear when obtaining ultrasound images, it has been shown that this imaging technique is more advantageous for tumor quantifications in mice compared to other imaging techniques such as computed tomography (CT) imaging and bioluminescence imaging (BLI). Researchers can investigate novel therapeutic targets using this technique by comparing lung tumor initiation and progression between different groups of mice.

This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.

超声成像对小鼠肺肿瘤进展的检测

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

人类乳腺上皮细胞致癌转化的体外评价

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Monitoring Breast Cancer Growth and Metastatic Colony Formation in Mice using Bioluminescence

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several animal models have been generated, including the widely used orthotopic mouse models, where cancer cells are injected into the mammary fat pad. However, these models cannot help monitor tumor growth kinetics and metastatic colonization. Cutting-edge tools to monitor cancer cells in real time in mice will significantly advance the understanding of tumor biology. 
Here, breast cancer cell lines stably expressing luciferase and green fluorescent protein (GFP) were established. Specifically, this technique contains two sequential steps initiated by measuring the luciferase activity in vitro and followed by the implantation of the cancer cells into mammary fat pads of nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice. After the injection, both the tumor growth and metastatic colonization are monitored in real time by the noninvasive bioluminescence imaging system. Then, the quantification of GFP-expressing metastases in the lungs will be examined by fluorescence microscopy to validate the observed bioluminescence results. This sophisticated system combining luciferase and fluorescence-based detection tools evaluates cancer metastasis in vivo, which has great potential for use in breast cancer therapeutics and disease management.

Here, we describe a noninvasive monitoring method involving luciferase and green fluorescent protein expression in various breast cancer cell lines. This protocol provides a technique to monitor tumor formation and metastatic colonization in real time in mice.

使用生物发光监测小鼠乳腺癌生长和转移集落形成

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several ...

Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction with other molecules, including other proteins or nucleic acids. This work presents an easily implementable protocol for quantifying arginine and its derivatives, including asymmetric and symmetric dimethylarginine (ADMA and SDMA, respectively) and monomethyl arginine (MMA). Following protein isolation from biological body fluids, tissues, or cell lysates, a simple method for homogenization, precipitation of proteins, and protein hydrolysis is described. Since the hydrolysates contain many other components, such as other amino acids, lipids, and nucleic acids, a purification step using solid-phase extraction (SPE) is essential. SPE can either be performed manually using centrifuges or a pipetting robot. The sensitivity for ADMA using the current protocol is about 100 nmol/L. The upper limit of detection for arginine is 3 mmol/L due to SPE saturation. In summary, this protocol describes a robust method, which spans from biological sample preparation to NMR-based detection, providing valuable hints and pitfalls for successful work when studying the arginine methylome.

The present protocol describes the preparation and quantitative measurement of free and protein-bound arginine and methyl-arginines by 1H-NMR spectroscopy.

用核磁共振波谱探索精氨酸甲基组

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction ...

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, senescence is a fundamentally different machinery restraining&#160;propagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated &#946;-galactosidase (SA-&#946;-gal), the DNA synthesis marker 5-ethynyl-2&#8242;-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (&#947;H2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells.

Here we present a flow cytometry-based method for visualization and quantification of multiple senescence-associated markers in single cells.

基于同步成像和流式细胞术检测治疗诱导的衰老癌细胞中的多种荧光衰老标志物

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...