评估从3D多细胞肺肿瘤球体中分离的癌症相关成纤维细胞的线粒体健康
Summary
用肺腺癌细胞、成纤维细胞和单核细胞制备多细胞3D肿瘤球体，然后从这些球体中分离癌症相关成纤维细胞（CAF）。将分离的CAF与正常成纤维细胞进行比较，通过研究线粒体跨膜电位，活性氧和酶活性来评估线粒体健康状况。
Abstract
癌症相关成纤维细胞（CAF）是肿瘤微环境中最丰富的基质细胞之一，可促进肿瘤生长和进展。肿瘤微环境中的复杂性，包括肿瘤分泌组、低度炎症、缺氧和氧化还原失衡，促进了异型相互作用，并允许无活性的常驻成纤维细胞转化为活性 CAF。CAF 在代谢上与正常成纤维细胞 （NF） 不同，因为它们具有更高的糖酵解活性，产生更高水平的活性氧 （ROS），并且过表达乳酸输出剂 MCT-4，导致线粒体通透性过渡孔 （MPTP） 的打开。这里描述了一种方法来分析从多细胞3D肿瘤球体中分离的活化CAF的线粒体健康，该球状体包括人肺腺癌细胞（A549），人单核细胞（THP-1）和人肺成纤维细胞（MRC5）。肿瘤球体以不同的时间间隔分解，并通过磁激活细胞分选分离出CAF。使用JC-1染料评估CAF的线粒体膜电位，通过2’，7′-二氯二氢荧光素二乙酸酯（DCFDA）染色产生ROS以及分离CAF中的酶活性。分析分离CAF的线粒体健康有助于更好地了解反向Warburg效应，也可用于研究CAF线粒体变化的后果，例如代谢通量和相应的肺癌异质性调控机制。因此，本研究提倡了解肿瘤 – 基质相互作用对线粒体健康的影响。它将提供一个平台来检查线粒体特异性候选药物对CAF作为肿瘤微环境中潜在疗法的有效性，从而防止CAF参与肺癌进展。
Introduction
实体瘤由肿瘤微环境（TME）引导的异质细胞群组成，然而，大多数细胞的起源尚未被发现。主要是基质和免疫细胞（成纤维细胞、内皮细胞、单核细胞、巨噬细胞、树突状细胞、B 细胞、T 细胞及其亚群）反映了肺癌、乳腺癌、肾癌和其他实体癌的肿瘤异质性1，2，3。了解每种亚型的起源及其转分化潜力对于开发针对这些癌症的先进疗法至关重要。由于肿瘤类型、部位、分期、样本量限制和患者特异性变异性，在人活检中对这种多样化细胞群的分析本身就面临着一些挑战4。因此，需要一个实验模型，该模型不仅可靠，而且可以模拟体内肿瘤状况，证明其是研究肿瘤-基质串扰及其参与疾病病理生理学的理想选择。
三维（3D）多细胞肿瘤球状体（MCTS）培养物是一种有利的 体外 肿瘤模型系统，因为它们与天然对应物相似。与2D细胞培养模型相比，MCTS可以更好地复制实体瘤的各个方面，包括其空间结构，生理反应，可溶性介质的释放，基因表达模式和耐药机制。此外，MCTS的一个主要优点是它可用于研究肿瘤异质性和肿瘤微环境（TME）。悬挂-下降法是开发和分析 MCTS5 最常用的工具。在这种方法中，带有培养基的不同细胞以液滴的形式悬浮，这允许其以相干的3D聚集体方式生长，并且易于检查。该技术很简单;它不需要很多细胞，并且消除了球状体发育所需的特殊底物（如琼脂糖）6。该方法的另一个优点在于其技术的可重复性。此外，该方法还用于共培养混合细胞群，例如内皮细胞和肿瘤细胞，以模拟早期肿瘤血管生成7。
本研究采用模拟肺肿瘤微环境的悬挂滴法制备了含有肺腺癌细胞、成纤维细胞和单核细胞的多细胞三维肺肿瘤球体。然后分离癌症相关成纤维细胞（CAF）群体以调查线粒体健康状况。开发这些微球背后的主要思想是分离CAF，因为微球中细胞之间的串扰可以将成纤维细胞转化为肌成纤维细胞样激活的CAF状态。其次，这项研究也可能描述异常的ROS产生和线粒体功能障碍如何驱动正常的成纤维细胞走向更具侵略性的CAF表型。结果发现，在肿瘤球体内组装的成纤维细胞具有肌成纤维细胞特征，ROS活性增加，代谢基因表达诱导增加。该协议强调了肿瘤微环境在激活CAF中的重要性，并且可以成为 体外 生成和研究CAF表型特征的优秀模型。
Protocol
Representative Results
Discussion
本研究介绍了使用改进的悬挂滴法开发包括肿瘤细胞、基质细胞群（即成纤维细胞）和免疫细胞群（即单核细胞）的多细胞肿瘤球体。成纤维细胞和单核细胞/巨噬细胞是构成肿瘤微环境（TME）的最重要群体之一，它们的存在通常与患者预后不良有关16。当存在于TME中时，成纤维细胞发生转化，表现出特定的癌症相关成纤维细胞（CAF）表型，因为肿瘤微环境线索17</sup…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了印度塞尔维亚妇女卓越奖项目（SB/WEA-02/2017）和印度塞尔维亚-早期职业研究奖项目（ECR/2017/000892）的支持。作者LA和SR承认IIT Ropar和MHRD的研究奖学金。MK感谢ICMR的研究奖学金。
Materials
| Antibodies | |||
| APC anti-human α-SMA | R&D systems | Cat# IC1420A | |
| Anti-fibroblast microbeads | Miltenyi Biotec | Cat# 130-050-601 | |
| Cell lines | |||
| A549 lung adenocarcinoma cells | NCCS Pune | – | |
| MRC-5 fetal lung fibroblasts | ATCC | CCL-171 | |
| THP-1 Human monocytes | NCCS Pune | – | |
| Chemicals | |||
| BSA | Himedia | Cat# 9048-46-8 | |
| 2,6-dichloroindophenol (DCPIP) | SRL | Cat# 55287 | |
| Calcein-AM | Thermo Fisher Scientific | Cat# C3099 | |
| DAPI | Thermo Fisher Scientific | Cat# D1306 | |
| DCFDA | Sigma | Cat# D6883 | |
| DMEM | Gibco | Cat# 11995073 | |
| DPBS | Gibco | Cat# 14190-144 | |
| EDTA | Thermo fisher scientific | Cat# 17892 | |
| EGTA | SRL | Cat# 62858 | |
| EZcoun Lactate Dehydrogenase Cell Assay Kit | HiMedia | Cat# CCK036 | |
| FBS | Gibco | Cat# 10082147 | |
| Halt Protease and Phosphatase Inhibitor Cocktail (100X) | Thermo Fisher Scientific | Cat# 87786 | |
| HEPES | Thermo Fisher Scientific | Cat# 15630080 | |
| Horse heart Cytochrome c | SRL | Cat# 81551 | |
| Image-iT Red hypoxia reagent | Thermo Fisher Scientific | Cat# H10498 | |
| JC-1 Dye | Thermo Fisher Scientific | Cat# T3168 | |
| KCl | Merck | Cat# P9541 | |
| MgCl2 | Merck | Cat# M8266 | |
| MOPS | Thermo Fisher Scientific | Cat# 69824 | |
| Nacl | Sigma-Aldrich | Cat# S9888 | |
| NADH MB Grade | SRL | Cat# 54941 | |
| NP-40 | Thermo Fisher Scientific | Cat# 85124 | |
| Penicillin/Streptomycin | Gibco | Cat# 15140122 | |
| Phenazine methosulfate (PMS) | SRL | Cat# 55782 | |
| Propidium iodide | Thermo fisher scientific | Cat# P1304MP | |
| RPMI 1640 | Gibco | Cat# 11875093 | |
| Single Cell Lysis Kit | Thermo Fisher Scientific | Cat# 4458235 | |
| Sodium ascorbate | Merck | Cat# A7631 | |
| Sodium cyanide | Sigma | Cat# 205222 | |
| Sodium Deoxycholate | Thermo Fisher Scientific | Cat# 89904 | |
| Sodium dodecyl sulphate | Sigma-Aldrich | Cat# L3771 | |
| Sodium succinate hexahydrate | SRL | Cat# 36313 | |
| Sucrose | Sigma | Cat# S0389 | |
| SuperScript VILO cDNA synthesis kit | Thermo Fisher Scientific | Cat# 11754-050 | |
| Triton X-100 | Sigma | Cat# T8787 | |
| Trypsin 0.25% EDTA | Gibco | Cat# 25200072 | |
| Universal SYBR Green Supermix | BIO-RAD | Cat# 172-5124 | |
| Plasticware | |||
| MACS LS Columns | Miltenyi Biotec | Cat# 130-042-401 | |
| Equipment | |||
| Countess II FL Automated Cell Counter | Thermo Fisher Scientific | Cat# AMQAF1000 | |
| EVOS XL core imaging system | Thermo Fisher Scientific | Serial Number F0518-1727-0191 | |
| LAS X software | Leica Microsystems | ||
| Leica fluorescent inverted microscope | s | DMi8 automated S/N 424150) | |
| Midi MACS separator | Miltenyi Biotec | Cat# 130-042-302 |
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