淋巴结冷冻科肿瘤细胞粘附的定量
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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 …
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 15 mL Conical Tubes | Corning | 352096 | |
| 2-propanol | Merck | 109634 | |
| Benchtop Laminar Flow | Esco Cell Culture | ||
| Bin for Disc | Leica | 14020139126 | |
| Bovine Serum Albumin | Sigma-Aldrich | A9647-100 | |
| Cell culture flask T-25 cm2 | Corning | 430372 | |
| Cryostat | Leica | CM1860 UV | |
| Cryostat-Brush with magnet | Leica | 14018340426 | |
| DiIC18 Cell Traker Dye | Molecular Probes | V-22885 | |
| Fetal Bovine Serum (FBS) | Life Technologies | 12657-029 | |
| Fluorescence microscope | Nikon Eclipse 80 | ||
| Forma Series II CO2 incubator | Thermo Scientific | ||
| Formaldehyde | Sigma-Aldrich | 252549 | |
| High Profile Disposable Razor | Leica | 14035838926 | |
| Incubation Cube (IHC) | KASVI | K560030 | |
| Inverted microscope | Olympus | CKX31 | |
| Isofluran 100 mL | Cristália | ||
| Liquid Bloquer Super Pap Pen | Abcam, Life Science Reagents | ab2601 | |
| Optimal Cutting Temperature "OCT" compound | Sakura | 4583 | |
| Phosphate-buffered Saline (PBS) | Life Technologies | 70011-044 | |
| Poly-L-lysine | Sigma-Aldrich | P8920 | |
| RPMI | Gibco | 31800-022 | |
| Serological Pipettes 1 mL | Jet Biofil | GSP010001 | |
| Serological Pipettes 10 mL | Jet Biofil | GSP010010 | |
| Serological Pipettes 2 mL | Jet Biofil | GSP010002 | |
| Serological Pipettes 5 mL | Jet Biofil | GSP010005 | |
| Serological Pipettes 50 mL | Jet Biofil | GSP010050 | |
| Serological Pipettor Easypet 3 | Eppendorf | ||
| Tissue-Tek cryomold | Sakura | 4557 | |
| Trypan Blue 0.4% | Invitrogen | T10282 | |
| Trypsin | Instituto Adolfo Lutz | ATV |
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