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Biologie

侵入斑马鱼大脑的胶质母细胞瘤细胞微管动力学的实时成像

Published: July 29, 2022
doi:
10.3791/64093
, ,
1Cell Polarity, Migration and Cancer Unit,Université Paris-Cité, Institut Pasteur, Équipe Labellisée Ligue Contre le Cancer

Summary

我们报告了一种允许对侵入脊椎动物脑组织的胶质母细胞瘤（GBM）细胞中的微管动力学进行实时成像的技术。将荧光标记的GBM细胞的原位注射耦合到具有高分辨率活体成像的透明斑马鱼脑中，可以在 原位 癌症侵袭期间测量细胞骨架动力学。

Abstract

胶质母细胞瘤（GBM）在实际人群中中生存时间令人沮丧 – 在6至15个月之间 – 是最具破坏性的恶性脑肿瘤。治疗失败主要是由于GBM细胞的侵袭性，这说明了需要更好地了解GBM运动特性。为了研究支持GBM侵袭的分子机制，需要新的生理模型，能够深入表征侵袭过程中的蛋白质动力学。这些观察结果将为发现阻断肿瘤浸润和改善患者预后的新靶点铺平道路。本文报道了斑马鱼大脑中GBM细胞的原位异种移植如何允许亚细胞活体活体成像。专注于微管（MTs），我们描述了GBM细胞中MT标记的程序，在受精后3天（dpf）斑马鱼幼虫的透明大脑中微注射GBM细胞，在播散异种移植物中对MT进行活体成像，改变MT动力学以评估它们在GBM入侵期间的作用，并分析获得的数据。

Introduction

细胞运动是一个刻板的过程，需要极性轴的建立和产生力的细胞骨架重排。肌动蛋白聚合及其与肌球蛋白的关联被认为是细胞运动所需的突出和收缩力的主要贡献者1。微管被认为是迁移过程中细胞极化和定向持久性的主要参与者2。近年来，MTs也被证明可以产生和稳定突起，以支持3D3细胞侵袭期间的机械压缩力。最近，MTs直接参与了粘连和机械敏感迁移的机械转导4。表征MT-plus末端动力学的动态不稳定性由聚合（生长）和解聚（收缩）的重复阶段组成，这些阶段由大量的微管结合蛋白和细胞内信号级联控制，例如由RHO-GTPase5，6，7控制的那些。MT网络在细胞迁移和侵袭中的作用使得MT动力学的研究成为更好地了解免疫细胞归巢，伤口愈合和癌症侵袭机制的关键因素。

癌细胞逃离原发性肿瘤核心、在组织中扩散并产生继发性肿瘤的能力是防止全球在 50 年前宣布的抗癌战争中取得成功的关键一步8，9.最大的障碍之一是了解癌细胞如何主动侵入组织。关键侵袭机制依赖于与控制非肿瘤细胞迁移相同的原理10。然而，癌细胞迁移特异性已经出现11，引发了对这种类型的迁移进行更好表征的需求。具体来说，由于肿瘤微环境似乎是癌症进展的关键参与者12，因此在相关的生理背景下观察和分析癌细胞侵袭对于揭示癌细胞播散的机制至关重要。

MT是癌症进展的核心，以维持增殖和侵袭。原 位 MT动力学的精确分析有助于识别两个过程中的MT改变剂（MTA）。MT动态会随着环境的变化而发生巨大变化。在 体外，当细胞以3D形式包埋在凝胶中时，用MT去稳定剂（如诺考达唑）处理可防止细胞突起形成，而对2D细胞迁移几乎没有影响13，14。尽管技术上具有挑战性，但活体成像的进步允许在癌细胞侵袭期间对MT动力学进行 体内 分析。例如，在小鼠皮下异种移植纤维肉瘤细胞中对MT的观察表明，肿瘤相关巨噬细胞影响肿瘤细胞中的MT动力学15。然而，这些小鼠模型涉及广泛的外科手术，并且对于不易接近的癌症（例如高度侵袭性脑肿瘤GBM）仍然不满意。

尽管15个月的平均生存时间令人沮丧16，但人们对GBM在脑实质内的传播方式或维持GBM细胞侵袭脑组织的关键分子元素知之甚少。小鼠原位异种移植（PDX）模型的改进和颅窗的建立为GBM细胞侵袭研究提供了新的前景17，18。然而，由于成像质量欠佳，该模型主要允许对浅表异种移植物进行纵向成像，并且迄今为止尚未成功用于研究细胞骨架蛋白的亚细胞成像。此外，在“3R”禁令减少使用啮齿动物并用低等脊椎动物取而代之之后，已经建立了替代模式。

利用在斑马鱼（Danio rerio）幼虫中观察到的原始免疫力，在鱼脑中原位注射GBM细胞19，20，21。在发育中的中脑心室附近注射概括了大部分人类GBM病理生理学21，并且观察到与人类血管共同选择相同的GBM侵袭首选模式22。由于鱼幼虫的透明度，该模型允许可视化GBM细胞从大多数GBM产生的脑室周围区域侵入大脑23。

由于MT对于体外GBM细胞侵袭至关重要24，25，因此需要更好地表征MT动力学并鉴定细胞侵袭期间的关键调节因子。然而，迄今为止，斑马鱼原位模型生成的数据尚未包括入侵过程中MT动力学的亚细胞分析。本文提供了一种方案来研究体内MT动力学并确定其在脑癌侵袭中的作用。在稳定的微管标记之后，GBM细胞在斑马鱼幼虫的大脑中以3dpf的速度显微注射，并在脑组织中的进展过程中以高时空分辨率实时成像。荧光MT的实时成像允许对MT加端动力学进行定性和定量分析。此外，该模型可以实时评估MTA对MT动力学和GBM细胞侵袭特性的影响。这种相对非侵入性的方案与一次处理的大量幼虫以及药物应用的便利性（在鱼水中）相结合，使该模型成为临床前测试的资产。

Protocol

动物实验是根据欧盟处理实验动物的准则进行的。所有协议均由巴斯德研究所动物实验伦理委员会 – CEEA 89 和法国研究与教育部批准（许可证 #01265.03）。在注射或实时成像过程中，动物在实验程序结束时 Tricaine.At 麻醉，它们被麻醉剂过量安乐死。有关此协议中使用的材料、设备和软件的详细信息，请参阅 材料表 。该协议的一般工作流程如图 1 所示。 <p class=…

Representative Results

为了分析MTs在体内GBM侵袭中的作用，我们在这里描述了通过慢病毒感染在GBM细胞中进行稳定MT标记的主要步骤，3 dpf斑马鱼幼虫中GBM细胞的原位异种移植，MT动力学的高分辨率活体成像，MTA处理及其对GBM侵袭的影响，以及MT动力学和体内侵袭的图像分析（图1）。 MT动态是通过沿着增长和收缩的MT构建kymograph来测量的（图3C），或?…

Discussion

以单细胞分辨率对肿瘤异种移植物进行成像很可能成为提高我们对GBM生物学理解的不可或缺的工具。小鼠PDX模型中的实时成像导致了关于GBM如何集体侵入脑组织的宝贵发现18。然而，迄今为止，时空分辨率还不足以揭示控制GBM侵袭的蛋白质的动力学。我们推断，通过将透明斑马鱼幼虫中GBM细胞的原位移植与高分辨率活体成像相结合，可以足够详细地分析细胞骨架蛋白（如MTs）以?…

Offenlegungen

The authors have nothing to disclose.

Acknowledgements

我们非常感谢P. Herbomel博士（法国巴斯德研究所）和他的实验室，特别是Valérie Briolat和Emma Colucci-Guyon为我们提供斑马鱼生产线和显微注射板的塑料模具，以及他们在斑马鱼实验程序方面的宝贵专业知识。我们非常感谢UtechS光子生物成像（C2RT，巴斯德研究所，由法国国家研究机构法国生物成像和ANR-10-INBS-04支持;对未来的投资）。这项工作得到了Ligue contre le cancer（EL2017。LNCC）、国家科学研究中心和巴斯德研究所，以及玛格丽特·米歇尔夫人和波尔凯先生的慷慨捐赠。

Materials

Glioblastoma cell culture
Foetal calf serum Eurobio CVFSVF00-01 Reagent
MEM NEAA Gibco 11140-050 Reagent
Modified Eagle's medium Eurobio CM1MEM18-01 Reagent
Penicillin–streptomycin Gibco 15140-122 Reagent
U-87 MG ECACC 89081402-1VL Cells
Lenitivirus production
BD FACSAria III BD bioscience Instrument
BD FACSDiva software v8.0 BD bioscience Software
HEK-293T Merck 12022001 Cells
pMD2.G Addgene Plasmid #12259 Reagent
psPAX2 Addgene Plasmid #12260 Reagent
Ultracentrifuge Optima XPN-80 Beckman Coulter Instrument
Cell passaging and staining
dPBS Gibco 14190-094 Chemical
Hoechst 34580 Sigma-Aldrich 63493 Chemical
Trypsin-EDTA (0,05%) Gibco 25300-054 Reagent
Zebrafish husbandry
Fluorescence stereomicroscope LEICA M165FC LEICA https://www.leica-microsystems.com/fr/produits/stereomicroscopes-et-macroscopes/informations-detaillees/leica-m165-fc/ Instrument
Methylene Blue hydrate Sigma-Aldrich M4159 Chemical
N-Phenylthiourea (PTU) Sigma-Aldrich P7629-25G Chemical
Transfer Pipettes fine tips Samco Scientific 232 Equipment
Transfer Pipettes Large Bulb3mL Samco Scientific 225 Equipment
Tricaine (Ethyl 3-aminobenzoate methanesulfonate) Sigma-Aldrich Cat#: A5040 Chemical
Volvic Source Water DUTSCHER DOMINIQUE SAS 999556 Reagent
Xenotransplantation
24-well plate TPP 92024 Equipment
Borosilicate glass capillaries (1.0 ODx0.58IDx150L mm) Harvard Apparatus (#30-0017 GC100-15 Equipment
CellTram oil vario microinjector Eppendorf 5176000.025 Instrument
Microloading pipet tips (Microloader) 20µL Eppendorf  5242956003 Equipment
Micromanipulator NARISHIGE https://products.narishige-group.com/group1/injection/english.html Equipment
Mineral Oil Sigma M8410-100ml Equipment
Stereomicroscope Olympus KL 2500 LCD Instrument
Universal capillary holder Eppendorf 5176190002 Equipment
Vertical Pipette puller KOPF (Roucaire) Model 720 Instrument
Intravital Imaging
3.5cm glass-bottom videoimaging dish MatTek Life Sciences, MA, USA P35G-1,5-14-C Equipment
Acquisition software: NIS-Elements-AR version 5.21 Nikon Software
Heat-Block Techne DRI-BLOCK DB-2D Equipment
Microscope head Nikon Ti2E Nikon Instrument
sCMOS camera Prime 95B Photometrics Instrument
sCMOS camera Orca Flash 4 Hammatsu Instrument
Ultrapure Low melting point agarose Invitrogen 16520-050 Chemical
Yokagawa CSU-W1 spinning disk unit Hammatsu Instrument
Drug Treatment
DMSO Sigma-Aldrich D2650-100ML Chemical
Nocodazole Sigma-Aldrich M1404-2MG Chemical
Image Analysis
Imaris 9.5.1 software Oxford Instruments Software
ImarisFileConverter 9.5.1 Oxford Instruments Software
DOWNLOAD MATERIALS LIST

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Tags

Microtubule DynamicsGlioblastoma CellsZebrafish BrainSubcellular Intravital ImagingBrain Cancer InvasionProtein AnalysisCell TransplantationMicroinjectionAnesthetized Larvae

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Peglion, F., Coumailleau, F., Etienne-Manneville, S. Live Imaging of Microtubule Dynamics in Glioblastoma Cells Invading the Zebrafish Brain. J. Vis. Exp. (185), e64093, doi:10.3791/64093 (2022).
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