Name: quantification of tumor cell adhesion in lymph node cryosections
Uploaded: 2020-02-09T12:00:00
Description: Watch this Scientific Journal Video about Quantification of Tumor Cell Adhesion in Lymph Node Cryosections at JoVE.com

我们感谢罗莎娜·德利马·帕加诺博士和阿纳·卡罗莱纳·皮涅罗·坎波斯博士的技术援助。这项工作得到了来自：FAPESP - 圣保罗研究基金会（2016/07463-4）和路德维希癌症研究所（LICR）的资助。

从因宫颈脱位而牺牲的健康成年威斯塔大鼠的新鲜尸体中找到了LN。我们遵循了NIH《实验室动物疼痛和痛苦指南》，所有程序都通过了西里奥-利巴尼亚医院研究与教育研究所伦理委员会和动物研究（CEUA P 2016-04）。
注：所有新鲜冷冻组织均被视为生物危害，应采取适当的生物安全预防措施处理。
1. 淋巴切除术和冷冻切除术
<ol><li>将成年威斯塔大鼠（180-2...

<ol>
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癌细胞淋巴系统传播需要各种复杂的细胞驱动事件。它们与原发性肿瘤的细胞分离和细胞外基质 （ECM） 架构的重塑一起启动，并通过进入哨点 LUN 的淋巴淋巴细胞进行持续化疗和主动迁移。如果癌细胞在局域网中粘附并存活，它们很容易扩散到其他辅助器官。在这里，我们描述了一种快速和低成本地分析肿瘤细胞和冷冻LUN之间特定粘合剂相互作用的简单方法。
在结构上，LAN ...

癌症转移是治疗失败的主要原因，也是癌症的主要威胁生命方面。正如130年前所假设的，当传播的肿瘤细胞（DTC，"种子"）的精英获得特定的生物能力，使他们能够避开原址，并在遥远的部位（"土壤"）1建立恶性生长时，转移传播的结果。最近，关于"种子和土壤"关系出现了几个新的概念，如诱导预转移利基（概念化为"种子"茁壮成长所需的"肥沃土壤"），由DTC自行播种原发性肿瘤，二级器官的"种子"休眠和转移2的平行进展模型。
对于大多数固体恶性肿瘤，DTC可以驻留并检测在许多间质器官，如骨髓和淋巴结（LUN）的患者有或没有临床转移的证据。由于肿瘤排出的LN是DTC区域传播的第一个位置，LN状态是一个重要的预后指标，并且通常与辅助治疗决策3相关。对于一些肿瘤类型，LN状态和坏结局之间的相关性很强，包括头颈部4、5、乳腺6、前列腺7、肺8、胃9、结肠直肠10、11和甲状腺癌12。
LAN 是淋巴系统的小卵器官，覆盖有视网膜细胞，并封闭着淋巴血管。这些器官是绝对必要的免疫系统的功能13。LN作为免疫循环细胞的吸引平台，将淋巴细胞和抗原呈现细胞结合在一起14。然而，LN也吸引循环肿瘤细胞。几十年来，LUN被描绘成转移性肿瘤细胞的被动运输路线。然而，最近的研究表明，肿瘤细胞也可以通过化学战术（化学基因）和/或haptoin（细胞外基质元素）线索引导到LN，这些线索由淋巴内皮15分泌。例如，肿瘤细胞中CCR7受体的过度表达有助于转移性黑色素瘤细胞对肿瘤排泄的16的引导。此外，细胞外LN蛋白为循环肿瘤细胞的招募和存活提供了粘合支架17。事实上，肿瘤排泄的LN为DTC的播种提供了肥沃的土壤，通过特定的LN微环境信号18，DTC可以维持在增殖或休眠状态。这些LN驻留DTC的最终命运是有争议的;一些工作表明，这些细胞是转移性进展19的被动指标，而另一些则提出，它们更有可能是抵抗的创始者（通过自种原位）和/或作为转移的细胞储存库（传播"种子"三级癌症生长）20，21。最近，使用临床前模型，已经证明，这些LN驻DTC的一小部分积极侵入血管，进入血液循环和殖民肺21。
考虑到在LN中癌细胞的存在是癌症攻击性和侵入性的标志，在这项研究中，我们优化了Brodt22开发的经典方法，定量测量肿瘤细胞在体外对LUN的附着力。使用基于荧光的测定，使我们能够开发一种低成本、快速、敏感和环保（非放射性）协议，用于检测肿瘤细胞和LN冷冻细胞之间的胶粘剂变化。利用表达不同水平的NDRG4基因表达和大鼠LN冷冻节的MCF-7乳腺癌细胞，证明该方案在体外肿瘤细胞粘附与LN转移之间具有良好的相关性，在乳腺癌患者中观察到的LN转移24。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL Conical Tubes</td>
			<td>Corning</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>2-propanol</td>
			<td>Merck</td>
			<td>109634</td>
			<td></td>
		</tr>
		<tr>
			<td>Benchtop Laminar Flow</td>
			<td>Esco Cell Culture</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bin for Disc</td>
			<td>Leica</td>
			<td>14020139126</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A9647-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture flask T-25 cm2</td>
			<td>Corning</td>
			<td>430372</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM1860 UV</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat-Brush with magnet</td>
			<td>Leica</td>
			<td>14018340426</td>
			<td></td>
		</tr>
		<tr>
			<td>DiIC18 Cell Traker Dye</td>
			<td>Molecular Probes</td>
			<td>V-22885</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Life Technologies</td>
			<td>12657-029</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Nikon Eclipse 80</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forma Series II CO2 incubator</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>252549</td>
			<td></td>
		</tr>
		<tr>
			<td>High Profile Disposable Razor</td>
			<td>Leica</td>
			<td>14035838926</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubation Cube (IHC)</td>
			<td>KASVI</td>
			<td>K560030</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Olympus</td>
			<td>CKX31</td>
			<td></td>
		</tr>
		<tr>
			<td>Isofluran 100 mL</td>
			<td>Crist&#225;lia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid Bloquer Super Pap Pen</td>
			<td>Abcam, Life Science Reagents</td>
			<td>ab2601</td>
			<td></td>
		</tr>
		<tr>
			<td>Optimal Cutting Temperature &#34;OCT&#34; compound</td>
			<td>Sakura</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered Saline (PBS)</td>
			<td>Life Technologies</td>
			<td>70011-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P8920</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Gibco</td>
			<td>31800-022</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipettes 1 mL</td>
			<td>Jet Biofil</td>
			<td>GSP010001</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipettes 10 mL</td>
			<td>Jet Biofil</td>
			<td>GSP010010</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipettes 2 mL</td>
			<td>Jet Biofil</td>
			<td>GSP010002</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipettes 5 mL</td>
			<td>Jet Biofil</td>
			<td>GSP010005</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipettes 50 mL</td>
			<td>Jet Biofil</td>
			<td>GSP010050</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipettor Easypet 3</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek cryomold</td>
			<td>Sakura</td>
			<td>4557</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue 0.4%</td>
			<td>Invitrogen</td>
			<td>T10282</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Instituto Adolfo Lutz</td>
			<td>ATV</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of tumor cell adhesion in lymph node cryosections

肿瘤排出淋巴结（LUN）不仅仅是肿瘤产生的废物的过滤器。它们是不同类型的癌症患者中传播肿瘤细胞临时居住的最常见区域部位之一。这些LN驻留肿瘤细胞的检测是与预后不良和辅助治疗决策相关的重要生物标志物。最近的小鼠模型表明，LN驻留肿瘤细胞可能是远距离转移的恶性细胞的重要来源。量化肿瘤细胞对LN帕伦奇马的粘附性的能力是实验研究的一个关键指标，该指标侧重于识别与淋巴/转移传播相关的基因或信号通路。由于 LUN 是复杂的 3D 结构，根据截面的不同，在组织部分中具有各种外观和组合物，因此其基质很难以完全控制的方式在体外实验复制。在这里，我们描述了一种简单而廉价的方法，它允许将粘结肿瘤细胞量化到LN冷冻片。使用同一LN的串行部分，我们调整由Brodt开发的经典方法，使用非放射性标签，并直接计算每个LN表面积的粘附肿瘤细胞的数量。LN粘附肿瘤细胞通过光显微镜很容易识别，并通过荧光法进行确认，给出一个粘附指数，显示细胞结合亲和力到LN帕伦奇马，这是分子改变的分子改变的亲基结合与其相关的LN-ligand。

通过评估表达不同级别的NDRG4基因（称为NDRG4阳性和NDRG4阴性细胞）的红色荧光MCF-7乳腺癌细胞的LN粘合电电位，通过检查大鼠LN粘附肿瘤细胞的含量，在细胞表面24处的β1-integrin聚类的负调制器，来说明该测定。此协议的原始映像示例如图2所示。如图2B所示，附着细胞的形态呈形四舍五入，并且在整...

Watch this Scientific Journal Video about Quantification of Tumor Cell Adhesion in Lymph Node Cryosections at JoVE.com

Quantification of Tumor Cell Adhesion in Lymph Node Cryosections

在这里，我们描述了一种简单而廉价的方法，它允许将粘结肿瘤细胞量化到淋巴结（LN）冷冻片。LN粘附肿瘤细胞通过光显微镜很容易识别，并通过荧光法进行确认，提供粘附指数，揭示肿瘤细胞结合亲和力与LN帕伦奇马。

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 ...

转移的最常见部位是原始淋巴结。索马特斯可用于检测肿瘤细胞淋巴结部分在体外粘合的亲和力。这项技术的一个优点是，新鲜的淋巴结部分提供了原始组织的自然复杂性，这是不可能使用纯化蛋白质重新创建。这种方法对于分子肿瘤学研究可能很有用，这些研究寻求识别干扰肿瘤细胞在转移性靶器官（如淋巴结）粘附的基因或疗法。要在牺牲后三十分钟内收获淋巴结，请将成年 Wistar 大鼠放在室温下干净的解剖板上，用 70%异丙醇喷洒毛皮。使用灭菌器，抬起腹部皮肤，做一个内层皮肤切口，以暴露腹部内脏。提取肠道，直到胸腔和腹部淋巴结变得可见。使用钝尖剪刀，小心切除淋巴结，而不伤害潜在的上肠动脉，并放置淋巴结到含有5 mL无菌PBS的15 mL管。当所有淋巴结都收获后，将每个低温面向下放置一个淋巴结，并添加足够的最佳切温介质来覆盖组织。捕捉冷冻后，使用低温恒温器在 22 摄氏度下获取 5 到 8 微米厚的部分，并收集到玻璃显微镜幻灯片上。来自同一淋巴结连续切片的高质量冷冻对于最大限度地减少切片之间的差异和促进一致的细胞粘附率至关重要。对于肿瘤细胞标签，从适当阶段性细胞培养物中收获感兴趣的细胞，并在每 mL 浓度和无血清培养的 6 个细胞下重新悬浮细胞。将1 mL的细胞转移到一个新的15 mL锥形管中，并在37摄氏度下用每毫升DII（C18）2微克标记细胞10分钟，在5分钟内轻轻搅拌以避免细胞沉积。在孵育结束时，通过离心对细胞进行颗粒，用10 mL的新鲜无血清介质洗涤两次，以去除多余的染料。第二次洗涤后，将无血清介质每mL的1 x 10至6个细胞重新悬浮，并辅以0.1%的牛血清白蛋白浓度。将标记的肿瘤细胞转移到收获的淋巴结组织部分，在PBS中轻轻清洗两次，随后在室温下在新鲜PBS中再水化15分钟。在孵育结束时，在37摄氏度下，用2.5%牛血清白蛋白阻断任何非特异性结合，在37摄氏度下30分钟，然后使用棉签擦干滑梯。使用疏水笔在每个部分周围绘制一个屏障，并将 100 微升标记的细胞添加到每个包围的淋巴结上。在加湿室37摄氏度下一至两小时后，在PBS中去除任何不粘附细胞，并在室温下将剩余的粘附荧光细胞与PBS中的3.7%甲醛修复15分钟。为了量化粘合剂指数，使用荧光显微镜上的 10 倍目标获取明亮和红色荧光场中每个部分的单个 TIFF 图像。然后，在斐济打开图像之前，系统地命名并保存图像。设置比例后，使用多边形工具选择要量化的感兴趣区域。选择以量化淋巴结区域。数据以平方毫米表示。接下来，选择插件、分析、单元格计数器，然后单击要量化的照片。单击初始化并选择计数器类型。然后，单击照片中的单元格。淋巴结粘附指数将表示为每个淋巴结覆盖区域的粘附肿瘤细胞的数量。要初始化下一张照片，请打开下一张照片，然后重复演示的量化。正如观察的，粘附细胞的形态是圆润的，细胞在异质上分散在感兴趣的淋巴结中。与相应的NDRG4阳性细胞相比，NDRG4阴性乳腺癌细胞系淋巴结粘附指数高2至3倍。此过程可调整为辅助其他转移目标组织（如大脑或肺）的粘附率，以评估被感染肿瘤细胞的辅助性优先位点。请记住，新鲜和冷冻组织应被视为生物危害，并且应该使用适当的生物安全预防措施进行处理。

Research

JoVE Journal

Cancer Research

Lung Tumor Cell Recruitment Assay

肺肿瘤细胞招募分析

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

科研

癌症研究

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

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

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 ...

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

人地幔细胞淋巴瘤移植小鼠模型中肿瘤植入的研究

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

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

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 ...

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

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 ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

体内在肿瘤微环境中 pH 值、 pO2、氧化还原状态、磷酸盐和谷胱甘肽浓度的 EPR 评价

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

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

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 ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

数字聚合酶链反应法测定零星家族性腺瘤性息肉病患者的基因变异

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells