Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Summary

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Abstract

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Introduction

Astrocytomas are the most common primary brain tumor and glioblastoma (GBM), a grade IV astrocytoma, is the most common and aggressive subtype with a median survival of 12-15 months^1,2. Invasion of diffuse astrocytomas, particularly GBM, precludes complete surgical resection, limits the effectiveness of adjuvant therapies, and inevitably leads to post-treatment recurrence³. Patients initially present either with de novo (primary) GBM or with lower grade astrocytomas that inevitably progresses to (secondary) GBM⁴. GBM is genomically heterogeneous and characterized by mutually exclusive and co-occurring mutations in genes that govern thr....

Protocol

All animal studies were approved by the University of North Carolina Institutional Animal Care and Use Committee.

1. Culturing Cortical Astrocytes from Neonatal Mice

1. Preparation
   1. Crumple 2-3 delicate task tissues and place into the bottom of a flask containing 70% ethanol. Place dissecting scissors, curved forceps and 2 pairs of straight micro forceps into this flask. The tissues are used to avoid bending the micro forceps.
   2. Add 1 ml of HBSS to a 60 mm tissue culture dish. Prepare a separate dish for each animal and maintain dishes on ice.
   3. Anesthetize animals per institutional regulations.

Results

We developed an nGEM model system with cortical astrocytes and NSC harvested from neonatal GEM harboring floxed conditional oncogenic alleles that can be phenotypically characterized in vitro and in vivo (Figure 1). In order to investigate the consequences of oncogenic mutations specifically in cortical astrocytes in vitro, it is critical to first enrich for astrocytes. Cortical astrocyte harvests contain a mixture of microglia, astrocytes, oligodendrocytes, OPC, and neurons, b.......

Discussion

The most critical steps to ensure proper harvest and culture of cortical astrocytes are 1) to excise the cortex without taking tissue below the corpus callosum, 2) to remove the meninges, 3) to thoroughly dissociate the cells, and 4) to enrich for GFAP⁺ astrocytes. Although we used mechanical (shaking) and genetic (restriction of genetic recombination with an Ad5GFAPCre vector or utilization of a dominant transforming transgene (TgGZT₁₂₁) under GFAP promoter control) metho.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco's Modified Eagle Medium (DMEM) (1X)	Invitrogen	11995065	DMEM can also be purchased from other suppliers including GIBCOSigma and Cellgro
Fetal Bovine Serum, Regular	Cellgro	35-010-CV
Penicillin-Streptomycin	Invitrogen	15140122
Sharp-Pointed Dissecting Scissors	Fisher Scientific	08-395
Cartilage Thumb Forceps, Curved	Fisher Scientific	1631
Miltex 17-301 Style 1 Jeweler Style Forceps, Fine, 4	Miltex	17-301
Ethanol (200 proof)	Decon Labs	2710
Razor Blades	VWR	55411-050
Hanks' Balanced Salt Solution (HBSS) (1x), liquid	Invitrogen	14175-095
TrypLE Express (1x), Phenol Red	Invitrogen	12605-010	Other trypsin solutions are also suitable for cortical astrocyte havest and culture
Culture Dish, 60 x 15 mm	Thomas Scientific	9380H77
15 ml Tubes	BD Biosciences	352096
50 ml Tubes	BD Biosciences	352070
Adenovirus stock	Gene Transfer Vector Core, U. Iowa	Ad5CMVCre	Store in 5 µl aliquots at -80 °C. Hazardous. Use Bsl2 safety precautions
Sodium sulfate (Na2SO4)	Sigma-Aldrich	238597
Potassium sulfate (K2SO4)	Sigma-Aldrich	221325
Magnesium chloride (MgCl2)	Sigma-Aldrich	M8266
Calcium chloride (CaCl2)	Sigma-Aldrich	746495
HEPES potassium salt	Sigma-Aldrich	H0527
D-(+)- Glucose	Sigma-Aldrich	G8270
Phenol Red	Sigma-Aldrich	P3532
Sodium hydroxide (NaOH)	Sigma-Aldrich	S5881
Papain	Worthington	LS003127
L-Cysteine-HCl	Sigma-Aldrich	C1276
Syringe filter (0.22 µm pore size)	Millipore	SLGP033NS
Neurocult proliferation kit, mouse	Stemcell Technologies	5702	This kit contains the NeuroCult NSC Basal Medium and NeuroCult NSC supplement needed for NSC culture
0.2% Heparin solution	Stemcell Technologies	7980
EGF	Invitrogen	PMG8041
bFGF	Invitrogen	PHG0261
Hanks' Balanced Salt Solution (HBSS) (10x)	Invitrogen	14185-052
Magnesium sulfate (MgSO4)	Sigma-Aldrich	M7506
Sodium bicarbonate (NaHCO3)	Sigma-Aldrich	S5761
E-64	Sigma-Aldrich	E3132	Make 10 mM stock in DMSO, store at -20 °C
6-well Plates	Fisher Scientific	07-200-83
Cell strainer (40 µm pore size)	Corning	352340
Stem cell dissociation solution	Stemcell Technologies	5707	Alternatively, use gentle enzyme solutions such as Accutase
Methyl cellulose 15 cP	Sigma-Aldrich	M7027
Dulbecco's Modified Eagle's Media 2x	Millipore	SLM-202-B	For making 5% methyl cellulose solution
1.7 ml Snap Cap Microcentrifuge Tube	Corning	3620
Hamilton syringe, 250 µl LT no needle	Fisher Scientific	14-815-92
PB600-1 Antigen Dispenser	Hamilton	83700
Disposable 18 G needles	Fisher Scientific	NC9015638
27 ga 1/2" luer tip needle	Fisher Scientific	14-826-48
2,2,2-Tribromoethanol (Avertin)	Sigma-Aldrich	T48402
Betadine	Fisher Scientific	NC9386574
Puralube Opthalmic Ointment	Fisher Scientific	NC9689910
Model 900 Stereotaxic frame	Kopf Instruments
VETBOND	Fisher Scientific	NC9259532	Tissue adhesive
Lidocaine	ShopMedVet	RXLIDO-EPI
CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS)	Promega	G3580
Anti-Sox2	Millipore	AB5603
Polyclonal Rabbit Anti-Glial Fibrillary Acidic Protein (GFAP)	Dako	Z0334
IVIS Kinetic	PerkinElmer		For in vivo imaging
D-Luciferin - K+ Salt Bioluminescent Substrate	PerkinElmer	122796	For in vivo bioluminescence imaging
EdU Imaging Kit	Invitrogen	C10340
MSCV Luciferase PGK-hygro	Addgene	18782