小肠神经内分泌肿瘤类球体的建立与特征
Summary
神经内分泌肿瘤（NETs）来自神经峰的神经内分泌细胞。它们生长缓慢，对文化充满挑战。我们提出了一个替代策略，通过培养它们作为球体，从小肠中种植NET。这些球体有小肠NET标记，可用于药物测试。
Abstract
小肠神经内分泌肿瘤（SBNETs）是肠道肠色素细胞的罕见癌症。这一领域的研究是有限的，因为很少患者衍生的SBNET细胞系被生成。分化良好的SBNET细胞生长缓慢，难以繁殖。已经建立的少数细胞系不是现成的，在培养中一段时间后可能无法继续表达NET细胞的特性。产生新的细胞系可能需要很多年，因为SBNET细胞有很长的倍增时间，并且需要许多浓缩步骤来消除快速分裂的癌症相关成纤维细胞。为了克服这些限制，我们开发了一种方案，从手术切除的肿瘤中培养SBNET细胞，将其作为细胞外基质（ECM）中的球形体。ECM 形成一个三维矩阵，封装 SBNET 细胞，并模仿肿瘤微环境，允许 SBNET 细胞生长。在这里，我们描述了SBNET球体的生长速率，并描述了使用免疫荧光显微镜和免疫组织化学识别SBNET标记物的方法，以确认球体是神经内分泌肿瘤细胞。此外，我们使用SBNET球体来测试拉帕霉素的细胞毒性。
Introduction
小肠神经内分泌肿瘤（SBNETs）起源于小肠的肠色素细胞。虽然SBNET通常已知生长缓慢，但它们通常转移至肝脏1。虽然手术切除或肿瘤消融可以考虑在许多情况下，复发几乎是普遍的，因此，药物治疗在管理中起着重要的作用。已经投入了巨大的努力来产生新的SBNET细胞系用于药物检测。然而，收效甚微。只报告了6个SBNET细胞系（KRJ-I、CND2、GOT1、P-STS、L-STS、H-STS）。不幸的是，一个细胞系不再表示NET标记6和三个其他SBNET细胞系（KRJ-I，L-STS，H-STS）被确定为从转化淋巴细胞，而不是NET7派生。为了加速识别针对SBNET的药物，需要采用替代方法进行体外药物检测。
在这里，我们利用被切除的SBNET的可用性，并建立了一种方法，培养这些患者衍生的SBNET作为在ECM中生长的球体。本手稿的总体目标是将一种将 SBNET 培养为三维 （3D） 培养方法描述为一种三维 （3D） 培养方法，并概述通过免疫荧光染色和免疫组织化学来描述这些球体以保留 SBNET 标记的过程。
此外，我们演示了这些SBNET球体如何可用于测试拉帕霉素的效果，一种抗癌药物NET8。该协议背后的原理是开发一种在体外培养SBNET细胞的新方法，并将其用于药物测试。与建立SBNET细胞系的传统方法相比，该技术的优点是可以快速获得SBNET的3D培养物，并在3周内进行药物测试。SBNET球体可能被用作执行体外药物屏幕的模型，以识别SBNET患者的新药。由于SBNET细胞系并不广泛可用，SBNET球体的3D培养物可以作为研究SBNET的新体外模型，并可在该领域的科学家之间共享。
Protocol
所有使用人类神经内分泌肿瘤样本的实验都已获得爱荷华大学医院和诊所IRB委员会的批准（协议号199911057）。所有材料和设备的列表在材料表中描述。增长介质和关键解决方案的列表见表1。 1. 小肠神经内分泌肿瘤（SBNET）采集和细胞分离 从外科病理学核心确认肿瘤组织后，获取切除的患者SBNET样本。 将 SBNET 切成 5 mm 立方体，并储存在…
Representative Results
目前只有2个SBNET细胞系建立和出版2，3，4，5，它们不是现成的许多研究人员。在这里，我们建议将 SBNET 培养为 ECM 中的球体，并将其用作研究 SBNET 药物敏感性的替代模型。从转移到肝脏的SBNET患者衍生的肿瘤被收集，消化以释放SBNET细胞，并与液体ECM混合，以建立SBNET球体培养物（图1A…
Discussion
肿瘤3D培养物已成为临床前药物检测的宝贵资源。各种肿瘤有机体生物库最近已建立从乳腺癌和前列腺癌肿瘤16，17。在这项研究中，我们为培养SBNET作为球体提供了详细的方案，以及一种简单快捷的方法，通过免疫荧光和测试药物敏感性验证NET标记物的球体培养。根据我们的经验，SBNET球体可以在各种文化媒介中成长。它们在干细胞培?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了NIH授予P50 CA174521（J.R.豪和A.M.贝利齐）的支持。P.H. Ear 是 P50 CA174521 职业提升计划奖的获得者。
Materials
| Anti-rabbit FITC | Jackson ImmunoResearch | 11-095-152 | Secondary antibody couple to a green fluorophore |
| Antigen Retrieval Solution | Agilent Dako | S2367 | Solution at pH 9 for preparing slides for IHC |
| Autostainer Link 48 | Agilent Dako | Not Available | Automated system for antibody staining |
| Cell freezing container | Thermo Scientific | 5100-0001 | Container to for freezing cells |
| CellSence | Olympus | Version 1.18 | Computer software for using fluorescent microscope |
| Chromogranin A antibody | Abcam-45179 | RB-9003-PO | Antibodies for IF |
| Chromogranin A antibody (clone LK2H10) | Thermo Scientific | MA5-13096 | Antibodies for IHC |
| Collagenase | Sigma | C0130 | Enzyme for digesting tumor tissue |
| DMEM | Gibco | 11965-092 | Medium for tissue preparation |
| DMEM/F12 | Gibco | 11320-033 | Medium for organoid cultures |
| DMSO | Sigma | D8418 | Solvent for dissolving drug |
| DNAse | Sigma | DN25 | Enzyme for digesting tumor tissue |
| Ethidium Homodimer | Chemodex | CDX-E0012-T1E | DNA and RNA binding dye |
| FBS | Gibco | 16000044 | Reagent for culture media |
| Fluorescent microscope | Olympus | CKX35 | Microscope for taking pictures of SBENT spheroids |
| Glutamine | Gibco | A2916801 | Reagent for culture media |
| ImageJ | National Institutes of Health | Version 1.51 | Computer software for image analysis |
| Insulin | Sigma | I0516 | Reagent for culture media |
| Matrigel | Corning | 356235 | Matrix to embed and anchore organoids |
| Mounting medium (VECTASHIELD) | Vector Laboratories | H-1200 | Fixative for labelled-cells with a nuclear stain |
| Nicotinamide | Sigma | 72340 | Reagent for culture media |
| Paraformaldehyde | Electron Microscopy Sciences | 15710 | Reagent to fix cells |
| PEN/STREP | Gibco | 15140-122 | Reagent for culture media |
| PT Link | Agilent Dako | Not Available | Automated system to prepare slides for IHC staining |
| Rapamycin | Alfa Aesar | J62473 | Drug that can inhibit NET growth |
| Secondary antibodies for IHC | Agilent Dako | K8000 | Secondary antibodies for IHC using Polymer-based EnVision FLEX system |
| SSTR2 antibody | GeneScritp | A01591 | Antibodies for IF |
| SSTR2 antibody (clone UMB1) | Abcam | ab134152 | Antibodies for IHC |
| Synaptophysin antibody | Abcam | 32127 | Antibodies for IF |
| Synaptophysin antibody (clone DAK-SYNAP) | Agilent Dako | M7315 | Antibodies for IHC |
| TritonX | Mallinckrodt | 3555 KBGE | Reagent to permeablize cells |
| Y-2763 ROCK inhibitor | Adipogen | AG-CR1-3564-M005 | To improve SBNET spheroid viability after freeze thaw |
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Citar este artigo
Ear, P. H., Li, G., Wu, M., Abusada, E., Bellizzi, A. M., Howe, J. R. Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids. J. Vis. Exp. (152), e60303, doi:10.3791/60303 (2019).