Translational Orthotopic Models of Glioblastoma Multiforme

Summary

Here, we describe a preclinical orthotopic mouse model for GBM, established by intracranial injection of cells derived from genetically engineered mouse model tumors. This model displays the disease hallmarks of human GBM. For translational studies, the mouse brain tumor is tracked by in vivo MRI and histopathology.

Abstract

Genetically engineered mouse (GEM) models for human glioblastoma multiforme (GBM) are critical to understanding the development and progression of brain tumors. Unlike xenograft tumors, in GEMs, tumors arise in the native microenvironment in an immunocompetent mouse. However, the use of GBM GEMs in preclinical treatment studies is challenging due to long tumor latencies, heterogeneity in neoplasm frequency, and the timing of advanced grade tumor development. Mice induced via intracranial orthotopic injection are more tractable for preclinical studies, and retain features of the GEM tumors. We generated an orthotopic brain tumor model derived from a GEM model with Rb, Kras, and p53 aberrations (TRP), which develops GBM tumors displaying linear foci of necrosis by neoplastic cells, and dense vascularization analogous to human GBM. Cells derived from GEM GBM tumors are injected intracranially into wild-type, strain-matched recipient mice and reproduce grade IV tumors, therefore bypassing the long tumor latency period in GEM mice and allowing for the creation of large and reproducible cohorts for preclinical studies. The highly proliferative, invasive, and vascular features of the TRP GEM model for GBM are recapitulated in the orthotopic tumors, and histopathology markers reflect human GBM subgroups. Tumor growth is monitored by serial MRI scans. Due to the invasive nature of the intracranial tumors in immunocompetent models, carefully following the injection procedure outlined here is essential to prevent extracranial tumor growth.

Introduction

Glioblastoma (GBM; grade IV glioma) is the most common and malignant brain tumor, and current therapies are ineffective, resulting in a median survival of 15 months¹. Reliable and accurate preclinical models that represent the complex signaling pathways involved in brain tumor growth and pathogenesis are essential to expedite the progress in evaluating new therapeutic regimens for GBM. Mouse models in which human brain tumor cell lines are implanted subcutaneously in immunocompromised mice do not reflect the native immune environment of brain tumors, nor can they be used to evaluate the ability of therapeutics to cross the blood-brain barr....

Protocol

The study protocol described here was approved by the NCI at Frederick Animal Care and Use Committee. NCI-Frederick is accredited by AAALAC International and follows the Public Health Service Policy for the Care and Use of Laboratory Animals. Animal care was provided in accordance with the procedures outlined in the “Guide for Care and Use of Laboratory Animals (National Research Council, 2011; The National Academies Press, Washington D.C.).

1. Preparation of cells for injection

NOTE: Mouse brain tumor primary cells (MBRs) used for this model were originally isolated from tamoxifen induced TR....

Results

Mice injected with brain tumor cells should be monitored daily for signs of tumor growth such as seizures, ataxia, or weight loss. Brain tumor growth may also be monitored by MRI scanning at regular intervals. Weekly MRI scans allow the visualization of increasing tumor burden within the brain and tumor volume measurements (Figure 1C). In particular, TRP tumors exhibit aggressive growth, and 3D tumor volumes are measurable by MRI within 2 to 3 weeks post-intracranial injection (with an avera.......

Discussion

Preclinical models are essential for the evaluation of new therapeutic targets and novel treatment strategies in GBM. Genetically engineered mouse models for GBM have the advantage of tumor occurrence in the autochthonous site, but often with a long latency and unpredictable tumor growth¹³. The GEM model tumors exhibit a latency of 4-5 months, and the ideal time window for imaging, recruitment, and treatment is variable among individual mice. The orthotopic model has a well-established and tractab.......

Materials

Name	Company	Catalog Number	Comments
5% methylcellulose in 1X PBS, autoclaved	Millipore Sigma	M7027
1mL Tuberculin Syringe, slip tip	BD	309659
6" Cotton Tipped Applicators	Puritan	S-18991
Adjustable stage platform	David Kopf Instruments	Model 901
Aerosol Barrier Tips	Fisher Scientific	02-707-33
Alcohol Prep Pads Sterile, Large - 2.5 x 3 Inch	PDI	C69900
B6D2  mouse strain (C57Bl/6J x DBA/2J)	Jackson Laboratory	Jax #10006
Bone Wax	Surgical Specialties	901
Bupivacaine 0.25%	Henry Schein	6023287
BuprenorphineSR	ZooPharm	n/a
Clear Vinyl Tubing 1/8ID X 3/16OD	UDP	T10004001
CVS Lubricant Eye Ointment	CVS Pharmacy	247881
Disposable Scalpels, #10 blade	Scalpel Miltex	16-63810
Gas anesthesia machine with oxygen hook-up and anesthesia box	Somni Scientific	n/a	Investigator may use facility standard equipment
Gas anesthesia platform for mice	David Kopf Instruments	Model 923-B
GraphPad Prism	Graphpad	Prism      9      version 9.4.1
Hamilton 30 g needle, ½ “, small hub, point pst 3	Hamilton	Special Order
Hamilton precision microliter syringe, 1701 RN, no needle 10 µL	Hamilton	7653-01
Hot bead sterilizer with beads	Fine Science Tools	18000-45
Invitrogen Countess 3 Automated Cell Counter	Fisher Scientific	AMQAX2000
IsoFlurane	Piramal Critical Care	29404
Isopropyl Alcohol Prep Pads	PDI	C69900
ITK_SNAP (Version 36.X, 2011-present)	Penn Image Computing and Science Laboratory (PICSL) at the University of Pennsylvania, and the Scientific Computing and Imaging Institute (SCI) at the University of Utah
KOPF Small Animal Stereotaxic Instrument with digital readout console	David Kopf Instruments	Model 940
Masterflex Fitting, PVDF, Straight, Hose Barb Reducer, 1/4" ID x 1/8" ID	Masterflex	HV-30616-16
Mouse Heating Plate	David Kopf Instruments	PH HP-4M
Mouse Rectal Probe	David Kopf Instruments	PH RET-3-ISO
Nalgene Super Versi-Dry Surface Protectors	ThermoFisher Scientific	74000-00
P20 pipette	Gilson	F123600
Povidone Iodine Surgical Scrub	Dynarex	1415
Reflex 9 mm Wound Clip Applicator	Fine Science Tools	12031-09
Reflex 9 mm Wound Clip Remover	Fine Science Tools	12033-00
Reflex 9 mm Wound Clips	Fine Science Tools	12032-09
Semken forceps, curved	Fine Science Tools	11009-13
Temperature Controller	David Kopf Instruments	PH TCAT-2LV
Trypsin-EDTA (0.25%)	ThermoFisher Scientific	25200056
Tuberculin Syringe with 25g needle, slip tip	BD	309626
UltraMicroPump 3 with Micro2T Controller	World Precision Instruments	Model UMP3T