Live Imaging of Microtubule Dynamics in Glioblastoma Cells Invading the Zebrafish Brain

Summary

We report a technique permitting live imaging of microtubule dynamics in glioblastoma (GBM) cells invading a vertebrate brain tissue. Coupling the orthotopic injection of fluorescently tagged GBM cells into a transparent zebrafish brain with high-resolution intravital imaging allows the measurement of cytoskeleton dynamics during in situ cancer invasion.

Abstract

With a dismal median survival time in real populations-between 6 to 15 months-glioblastoma (GBM) is the most devastating malignant brain tumor. Treatment failure is mainly due to the invasiveness of GBM cells, which speaks for the need for a better understanding of GBM motile properties. To investigate the molecular mechanism supporting GBM invasion, new physiological models enabling in-depth characterization of protein dynamics during invasion are required. These observations would pave the way to the discovery of novel targets to block tumor infiltration and improve patient outcomes. This paper reports how an orthotopic xenograft of GBM cells in the zebrafish brain permits subcellular intravital live imaging. Focusing on microtubules (MTs), we describe a procedure for MT labeling in GBM cells, microinjecting GBM cells in the transparent brain of 3 days post fertilization (dpf) zebrafish larvae, intravital imaging of MTs in the disseminating xenografts, altering MT dynamics to assess their role during GBM invasion, and analyzing the acquired data.

Introduction

Cell motility is a stereotyped process requiring polarity axis establishment and force-generating cytoskeletal rearrangements. Actin polymerization and its association with myosin are recognized as the main contributors to protrusive and contractile forces required for cell movement¹. Microtubules are considered to be the main actors in cell polarization and directional persistence during migration². In recent years, MTs have also been shown to create and stabilize protrusions to support mechanocompressive forces during cell invasion in 3D³. More recently, MTs have been directly involved in mechan....

Protocol

Animal experiments were conducted according to European Union guidelines for the handling of laboratory animals. All protocols were approved by the Ethical Committee for Animal Experimentation of Institut Pasteur - CEEA 89 and the French Ministry of Research and Education (permit #01265.03). During injections or live imaging sessions, animals were anaesthetized with Tricaine.At the end of the experimental procedures, they were euthanized by anesthetic overdose. See the Table of Materials for details related to the materials, equipment, and software used in this protocol. The general workflow of the protocol is described in Figure ....

Results

To analyze the role played by MTs during in vivo GBM invasion, we describe here the major steps to perform stable MT labeling in GBM cells by lentiviral infection, orthotopic xenotransplantation of GBM cells in 3 dpf zebrafish larvae, high-resolution intravital imaging of MT dynamics, MTA treatment and its effects on GBM invasion, and image analysis of MT dynamics and in vivo invasion (Figure 1). MT dynamics are measured either by building kymographs along .......

Discussion

Imaging tumor xenografts at single-cell resolution is likely to become an indispensable tool to improve our understanding of GBM biology. Live imaging in mouse PDX models has led to valuable discoveries on how GBM collectively invades the brain tissue¹⁸. However, to date, the spatiotemporal resolution is not high enough to reveal the dynamics of proteins controlling GBM invasion. We reasoned that by coupling the orthotopic engrafting of GBM cells in transparent zebrafish larvae with high-resolutio.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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