Real-Time Monitoring of Human Glioma Cell Migration on Dorsal Root Ganglion Axon-Oligodendrocyte Co-Cultures

Summary

Here we present an ex-vivo mixed monolayer culture system for the study of human glioma cell (hGC) migration in real-time. This model provides the ability to observe interactions between hGCs and both myelinated and non-myelinated axons within a compartmentalized chamber.

Abstract

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better understand the molecular mechanisms underlying glioma cell migration, an ability to study the interaction between hGCs and axons within the tumor microenvironment is essential. In order to model this cellular interaction, we developed a mixed culture system consisting of hGCs and dorsal root ganglia (DRG) axon-oligodendrocyte co-cultures. DRG cultures were selected because they can be isolated efficiently and can form the long, extensive projections which are ideal for migration studies of this nature. Purified rat oligodendrocytes were then added on purified rat DRG axons and induced to myelinate. After confirming the formation of compact myelin, hGCs were finally added to the co-culture and their interactions with DRG axons and oligodendrocytes was monitored in real-time using time-lapse microscopy. Under these conditions, hGCs form tumor-like aggregate structures that express GFAP and Ki67, migrate along both myelinated and non-myelinated axonal tracks and interact with these axons through the formation of pseudopodia. Our ex vivo co-culture system can be used to identify novel cellular and molecular mechanisms of hGC migration and could potentially be used for in vitro drug efficacy testing.

Introduction

Glioblastoma is one of the most aggressive and lethal tumors of the human brain. The current standard of care includes surgical resection of the tumor followed by radiation¹ plus concomitant and adjuvant administration of temozolomide². Even with this multi-therapeutic approach, tumor recurrence is inevitable³. This is partly due to the extensive migratory nature of the tumor cells, which invade the brain parenchyma creating multiple finger-like projections within the brain⁴ that make complete resection unlikely.

In recent years, it has become ev....

Protocol

The protocols for collection, isolation, and propagation of patient-derived human glioma cells were approved by the IRB committee of Rhode Island Hospital. All animals were maintained according to the NIH Guide for the Care and Use of Laboratory Animals. All animal use protocols were approved by the Institutional Animal Care and Use Committee of Rhode Island Hospital.

1. Media and buffer preparations

1. Prepare 50 mL of Neurosphere Media: 1x Neuronal basal medium w/o vitamin A, 1x ser.......

Discussion

Migration studies for hGCs may be performed by using Boyden chamber systems or scratch assays. However, while these experiments fail to give any information regarding the interactions of tumor cells with other surrounding tissues, the present system can recapitulate GC interactions with myelinated and non-myelinated fibers. Furthermore, to study tumor formation and end-point migration, organotypic slice cultures of the rodent brain or in vivo implantation of glioma cells into the rodent brain or flank have previously bee.......

Materials

Name	Company	Catalog Number	Comments
100 mm Suspension Culture Dish	Corning	430591
2.5S NGF	ENVIGO	B.5025
60 mm Suspension Culture Dish	Corning	430589
ACK Lysing Buffer	Thermo Fisher	A1049201
Ammonium Hydroxide Solution	Fisher Scientific	A669-500	Concentrated
Animal-Free Recombinant Human EGF	Peprotech	AF-100-15
Animal-Free Recombinant Human FGF-basic (154 a.a.)	Peprotech	AF-100-18B
Anti-A2B5 MicroBeads, human, mouse, rat	Miltenyi Biotec	130-093-392
Antibiotic-Antimycotic (100X)	Thermo Fisher	15240062
AutoMACS Rinsing Solution (PBS, pH 7.2)	Miltenyi Biotec	130-091-222
B27 Supplement	Thermo Fisher	17504044
B27 Supplement, minus vitamin A	Thermo Fisher	12587001
Bacteriological Plate	BD Falcon	351029
Biotin	Sigma	B4639
BSA	Sigma	A9418
Campenot Chamber	Tyler Research	CAMP-10
Cell Culture Dish	Corning	430165	35mm X 10mm
Cell Strainer	BD Falcon	352350	70 uM, Nylon
Cell Strainer	BD Falcon	352340	30 uM, Nylon
Collagenase/Dispase	Roche	11097113001
Cultrex Rat Collagen I	Trevigen	3440-100-01
D-Glucose	Sigma	G5146
DMEM	Thermo Fisher	10313021
DNase I	Sigma	D7291
Dow Corning High-Vacuum Grease	Fisher Scientific	14-635-5D
Dumont #5 Forceps	Roboz	RS-5045
E16 Timed Pregnant Sprague Dawley Rat
EBSS	Sigma	E7510
EGTA	Sigma	E3889
FBS	Hyclone	SH30070.02
FUDR	Sigma	F0503
GlutaMAX Supplement	Thermo Fisher	35050061
Ham's F-12 Nutrient Mix	Thermo Fisher	11765054
HBSS	Thermo Fisher	14175095
Hemostatic Forceps	Roboz	RS-7035
Heparin Sodium Salt, 0.2% in PBS	Stem Cell Technologies	07980
Hypodermic Needle, 18G	BD	511097
Insulin-Transferrin-Selenium G	Thermo Fisher	41400045
L-Cysteine	Sigma	C7477
L-Glutamine	Thermo Fisher	25030081
Leibovitz's L-15 Medium	Thermo Fisher	11415064
MACS BSA Stock Solution	Miltenyi Biotec	130-091-376
MACS MultiStand	Miltenyi Biotec	130-042-303
MEM	Thermo Fisher	1190081
Mg2SO4	Sigma	M2643
MiniMACS Separator	Miltenyi Biotec	130-042-102
MS Columns plus tubes	Miltenyi Biotec	130-041-301
NAC	Sigma	A8199
NaHCO3	Sigma	S5761
Neurobasal Medium	Thermo Fisher	21103049
Neurobasal-A Medium	Thermo Fisher	10888022
Ordinary forceps
P2 Sprague Dawley Rat Pups
Papain	Worthington	LS003126
Penicillin-Streptomycin	Thermo Fisher	15140148
Pin Rake	Tyler Research	CAMP-PR
Progesterone	Sigma	P8783
StemPro Accutase Cell Dissociation Reagent	Thermo Fisher	A1110501
Syrine Grease Applicator	Tyler Research	CAMP-GLSS
Transferrin	Sigma	T2036
Uridine	Sigma	U3003