PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator

Summary

In the past, small animal irradiation was usually performed without the ability to target a well-delineated tumor volume. The goal was to mimic the treatment of human glioblastoma in rats. Using a small animal irradiation platform, we performed MRI-guided 3D conformal irradiation with PET-based sub-volume boosting in a preclinical setting.

Abstract

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without the ability to target a specific or well-delineated tumor volume. The delivery of radiation was achieved using fixed radiation sources or linear accelerators producing megavoltage (MV) X-rays. These devices are unable to achieve sub-millimeter precision required for small animals. Furthermore, the high doses delivered to healthy surrounding tissue hamper response assessment. To increase the translation between small animal studies and humans, our goal was to mimic the treatment of human glioblastoma in a rat model. To enable a more accurate irradiation in a preclinical setting, recently, precision image-guided small animal radiation research platforms were developed. Similar to human planning systems, treatment planning on these micro-irradiators is based on computed tomography (CT). However, low soft-tissue contrast on CT makes it very challenging to localize targets in certain tissues, such as the brain. Therefore, incorporating magnetic resonance imaging (MRI), which has excellent soft-tissue contrast compared to CT, would enable a more precise delineation of the target for irradiation. In the last decade also biological imaging techniques, such as positron emission tomography (PET) gained interest for radiation therapy treatment guidance. PET enables the visualization of e.g., glucose consumption, amino-acid transport, or hypoxia, present in the tumor. Targeting those highly proliferative or radio-resistant parts of the tumor with a higher dose could give a survival benefit. This hypothesis led to the introduction of the biological tumor volume (BTV), besides the conventional gross target volume (GTV), clinical target volume (CTV), and planned target volume (PTV).

At the preclinical imaging lab of Ghent University, a micro-irradiator, a small animal PET, and a 7 T small animal MRI are available. The goal was to incorporate MRI-guided irradiation and PET-guided sub-volume boosting in a glioblastoma rat model.

Introduction

High-grade glioma is the most common and most aggressive malignant brain tumor in adults with a median survival of 1 year despite current treatment modalities. The standard of care includes maximal surgical resection followed by combined external beam radiation therapy (RT) and temozolomide (TMZ), followed by maintenance TMZ^1,2,3. Since the introduction of TMZ now more than 15 years ago, no significant improvements have been made in the treatment of these tumors. Therefore, the implementation of new therapeutic strategies is urgent but should be first investigated in small an....

Protocol

The study was approved by the ethics committee for animal experiments (ECD 09/23 and ECD 12/28). All commercial details can be found in Table of Materials.

1. F98 GB Rat Cell Model

1. Culture the F98 GB cells, obtained from ATCC, in monolayers using Dulbecco's modified Eagle Medium, 10% calf serum, 1% penicillin, 1% streptomycin, 1% L-glutamine, and 0.1% amphotericin b, and place in a CO₂ incubator (5% CO₂ and 37 °C).
2. Inoculate the glioma cells in the brain of female Fischer F344 rats (body weight 170 g).
   1. Use sterile instruments and wear sterile gloves at all tim....

Results

To mimic the human treatment methodology for the irradiation of glioblastoma in a preclinical model, inclusion of MRI-guided radiotherapy was necessary. Using the PCTPS and the micro-irradiator interface we were able to irradiate F98 glioblastoma in rats with multiple conformal non-coplanar arcs targeting the contrast-enhanced region on T1-weighted MRI¹⁷. Rigid-body transformations in combination with a multi-modality bed were used for image registration between MR.......

Discussion

To achieve accurate irradiation of the glioblastoma tumor target in the rat brain, the micro-irradiator's on-board CT guidance was not sufficient. Brain tumors are hardly visible due to insufficient soft tissue contrast, even if contrast enhancement would be used. As such, MRI needs to be included to allow more precise irradiation. Using a sequential MR acquisition on a 7 T system and a CT acquisition on the micro-irradiator we were able to target the dose to the contrast-enhancing tumor tissue in the brain and calcu.......

Materials

Name	Company	Catalog Number	Comments
GB RAT model
F98 Glioblastoma cell line	ATCC	CRL-2397
Fischer F344/Ico crl Rats	Charles River	N/A	http://www.criver.com/products-services/basic-research/find-a-model/fischer-344-rat
Micropump system	World Precision Instruments	UMP3	Micro 4: https://www.wpiinc.com/products/top-products/make-selection-ump3-ultramicropump/#tabs-1
Stereotactic frame	Kopf	902	Model 902 Dual Small Animal Stereotaxic frame
diamant drill	Velleman	VTHD02	https://www.velleman.eu/products/view/?id=370450
Bone wax	Aesculap	1029754	https://www.aesculapusa.com/products/wound-closure/hemostatic-bone-wax
Insulin syringe Microfine	Beckton-Dickinson	320924	1 mL, 29G
InfraPhil IR lamp	Philips	HP3616/01
Ethilon	Ethicon	662G/662H	FS-2, 4-0, 3/8, 19 mm
Name	Company	Catalog Number	Comments
Cell culture
DMEM	Invitrogen	14040-091
Penicilline-streptomycine	Invitrogen	15140-148
L-glutamine	Invitrogen	25030-032
Fungizone	Invitrogen	15290-018
Trypsin-EDTA	Invitrogen	25300-062
PBS	Invitrogen	14040-224
Falcons	Thermo Scientific	178883	175 cm2 nunclon surface, disposables for cell culture with filter caps
Cell freezing medium	Sigma-aldrich	C6164	Cell Freezing Medium-DMSO, sterile-filtered, suitable for cell culture, endotoxin tested
Name	Company	Catalog Number	Comments
Animal irradiation
Micro-irradiator	X-strahl	SARRP
software for irradiation	X-strahl	MuriPlan	pre-clinical treatment planning system (PCTPS), version 2.0.5.
Name	Company	Catalog Number	Comments
Small animal PET
microPET system possibility 1	Molecubes	B-Cube	http://www.molecubes.com/b-cube/
microPET system possibility 2	TriFoil Imaging, Northridge CA	FLEX Triumph II	http://www.trifoilimaging.com
PET tracers	In-house made		18F-FDG, 18F-FET, 18F-FAZA, 18F-Choline
Name	Company	Catalog Number	Comments
Small animal MRI
microMRI system	Bruker Biospin	Pharmascan 70/16	https://www.bruker.com/products/mr/preclinical-mri/pharmascan/overview.html
Dotarem contrast agent	Guerbet		MRI contrast agent, Dotarem 0,5 mmol/ml
rat whole body transmitter coil	Rapid Biomedical	V-HLS-070
rat brain surface coil	Rapid Biomedical	P-H02LE-070
Water-based heating unit	Bruker Biospin	MT0125
30 G Needle for IV injection	Beckton-Dickinson	305128	30 G
PE 10 tubing (60 cm/injection)	Instech laboratories, Inc	BTPE-10	BTPE-10, polyethylene tubing 0.011 x .024 in (0.28 x 60 mm), non sterile, 30 m (98 ft) spool, Instech laboratories, Inc Plymouth meeting PA USA- (800) 443-4227- http://www.instechlabs.com
non-heparinised micro haematocrit capillaries	GMBH	7493 21	these capillaries are filled with water to create markers visible on MRI and CT
Name	Company	Catalog Number	Comments
Consumables
isoflurane: Isoflo	Zoetis	B506	Anaesthesia
ketamine: Ketamidor	Ecuphar		Anaesthesia
xylazine: Sedaxyl	Codifar NV		Anaesthesia
catheter	Terumo	Versatus-W	26G
Temozolomide	Sigma-aldrich	T2577-100MG	chemotherapy
DMSO	Sigma-aldrich	276855-100ML
Insulin syringe Microfine	Beckton-Dickinson	320924	1 mL, 29G
Name	Company	Catalog Number	Comments
Image analysis
PMOD software	PMOD technologies LLC	PFUS (fusion tool)	biomedical image quantification software (BIQS), version 3.405, https://www.pmod.com/web/?portfolio=22-image-processing-pfus
Name	Company	Catalog Number	Comments
Anesthesia-equipment
Anesthetic movabe unit	ASA LTD	ASA 0039	ASA LTD, 5 valley road, Keighley, BD21 4LZ
Oxygen generator	Veterinary technics Int.	7F-3	BDO-Medipass, Ijmuiden