The authors would like to thank Stichting Luka Hemelaere and Soroptimist International for supporting this work.

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
<ol>
	<li>Culture the F98 GB cells, obtained from ATCC, in monolayers using Dulbecco&#39;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 &#176;C).</li>
	<li>Inoculate the glioma cells in the brain of female Fischer F344 rats (body weight 170 g).
	<ol>
		<li>Use sterile instruments and wear sterile gloves at all times.</li>
		<li>Anesthetize the rats by injecting a mixture of 74 mg/kg ketamine and 11 mg/kg xylazine intrapertioneally (IP) with an insulin syringe (1 mL, 29 G). Confirm the anesthetization by the absence of response to the withdrawal reflex of the limb. Immobilize the rats in a stereotactic device using fixation points for nose and ears. Place a carbomer eye gel to prevent dryness of the eyes while under anesthesia.</li>
		<li>Shave the rat from eye level to the back of the skull and disinfect the skin with povidone-iodine.</li>
		<li>Expose the skull through a midline scalp incision of 2 cm, and make a 1 mm hole (diamond drill) 2 mm posterior and 2.5 mm lateral to the bregma in the right frontal hemisphere.</li>
		<li>Insert a stereotactically guided insulin needle (29 G) and inject 5 &#181;L cell suspension (20,000 F98 GB cells) 3 mm deep using an microsyringe pump controller (settings: inject (I50), rate 1 nL/s (001 SDN)).</li>
		<li>Withdraw the syringe slowly and close the incision with bone wax. Suture the skin and disinfect with povidone-iodine.</li>
		<li>Stabilize the body temperature of the animal post-surgery using a red lamp. Monitor the awakening of the rat until it has regained sufficient consciousness to maintain sternal recumbency. Do not return the animal to the company of other animals until fully recovered. Keep all animals under environmentally controlled conditions (12 h normal light/dark cycles, 20-24 &#176;C, and 40-70% relative humidity) with food and water ad libitum. Make sure to follow the animals closely by monitoring their body weight, food, water intake, and their activity and normal behavior. Use a lethal dose of pentobarbital sodium to euthanize the animals (160 mg/kg) if a decline of 20% body weight is observed or when the normal behavior severely deteriorates (e.g., lack of grooming).</li>
	</ol>
	</li>
</ol>
2. Confirmation of Tumor Growth
NOTE: Evaluate tumor growth 8 days post-inoculation using T2-weighted MRI, dynamic contrast-enhanced MRI (DCE-MRI), and contrast-enhanced T1-weighted MRI. When the tumor reaches a size of 2.5 x 2.5 x 2.5 mm3, select the rat for therapy.
<ol>
	<li>First, connect a 30 G needle to a 60 cm long tube, which is placed intravenously in the lateral tail vein. Anesthetize the rats through a nose cone with 2% isoflurane mixed with oxygen (0.3 L/min). Confirm anesthetization when the rats do not respond to the withdrawal reflex of the limb. Cover the rats with a heated blanket and place them in the MRI bed. Use a carbomer eye gel to prevent dryness.</li>
	<li>Place the bed in the holder with a fixed rat brain surface coil, and position the bed in a 72 mm rat whole body transmitter coil.</li>
	<li>Perform a localizer scan followed by a T2-weighted spin-echo scan to assess tumor growth. T2-MRI sequence details: TR/TE 3661/37.1 ms, 109 &#181;m isotropic in-plane resolution, slice thickness 600 &#181;m, 4 averages, TA 9 min 45 s.</li>
	<li>If tumor is confirmed on the T2-weighted acquisition, inject a gadolinium-containing contrast agent into the intravenously placed tubing (MRI contrast agent; 0.4 mL/kg) 30 s after the start of the DCE-MRI acquisition. Acquire DCE-MRI during 12 min using a fast-low angle shot (FLASH) sequence in a single slice (1 mm slice thickness). Use an in-plane spatial resolution of (312 &#181;m2) and a temporal resolution of 1.34 s.</li>
	<li>Using the image sequence analysis tool, select a region of interest (ROI) within the suspected tumor region to plot the signal intensity over time. Subsequently, analyze the shape of the resulting DCE curve to confirm the presence of glioblastoma (Figure 3).</li>
	<li>Finally, acquire a contrast-enhanced T1-weighted spin-echo sequence. T1-MRI sequence details: TR/TE 1539/9.7 ms, 117 &#181;m isotropic in-plane resolution, slice thickness 600 &#181;m, 3 averages, TA 4 min 15 s. Typical contrast-enhanced T1-weighted MR images are shown in Figure 2.</li>
	<li>After finalizing the T1-weighted sequence, the animal can wake up under continuous supervision, until it regains full conciousness.</li>
</ol>
3. Multimodality Imaging for Target Volume Selection
NOTE: To be able to perform MRI-guided 3D conformal irradiation of the F98 GB rat model with PET-guided sub-volume boosting, 3 imaging modalities need to be performed. First, inject the radiotracer, then perform MRI during tracer uptake, subsequently perform a static PET acquisition and a treatment planning CT.
<ol>
	<li>Anesthetize the animals using a nose cone with 2% isoflurane mixed with oxygen (0.3 L/min). Confirm anesthetization when the rats do not respond to the withdrawal reflex of the limb. Use a carbomer eye gel to prevent dryness while under anesthesia.</li>
	<li>Insert a catheter (26 G) into the tail vein, enabling the injection of 37 MBq of PET radioactive tracer dissolved in 200 &#181;L saline. Inject either 18F-FET or 18F-FAZA, 30 min or 2 h before PET acquisition, respectively.</li>
	<li>Inject MRI contrast agent (0.4 mL/kg) intravenously in the tail vein using the catheter 15 min before PET acquisition.</li>
	<li>Place the rats on an in-house made multimodality bed and secure using hook-and-loop fasteners, maintaining a fixed position during imaging and micro-irradiation (Figure 1).</li>
	<li>Fix three multimodality markers (capillaries filled with water) underneath, above, and on the right side of the skull. Place the rat, still fixed on the multimodality bed, in the animal holder of the MRI scanner, fix the rat brain surface coil and position this set-up in a 72 mm rat whole-body transmitter coil. Perform a localizer scan followed by a contrast-enhanced T1-weighted spin-echo sequence.</li>
	<li>Transport the animal to perform a 18F-FET or 18F-FAZA PET acquisition. Acquire a 30 min static PET scan in list-mode. Scan should be acquired either 30 min after 18F-FET injection or 2 h after 18F-FAZA injection. Reconstruct all PET scans into a 200 &#215; 200 &#215; 64 matrix by a 2D Maximum Likelihood Expectation Maximization (MLEM) algorithm using 60 iterations and a voxel size of 0.5 &#215; 0.5 &#215; 1.157 mm.</li>
	<li>Place the animal, still fixed on the multimodality bed, on a plastic holder secured onto the four-axis robotic positioning table of the micro-irradiator. Perform a high-resolution treatment planning CT scan using an aluminum filter of 1 mm and a 20 x 20 cm (1,024 x 1,024 pixel) amorphous Si flat panel detector. Reconstruct the CT images with an isotropic voxel size of 0.2 mm. Fix the tube voltage and tube current at 70 kV and 0.4 mA, respectively. Acquire a total of 360 projections over 360 &#176;.</li>
</ol>
4. RT Treatment Planning
<ol>
	<li>Use the pre-clinical treatment planning system (PCTPS) for treatment planning. Import the planning CT into the PCTPS and manually segment this CT image into three different tissue classes: bone, soft tissue, and air. This manual segmentation is based on defining three different grey-value thresholds on the planning CT. These manually selected grey-value thresholds should be chosen such that air in the brain is absent and that bone thickness of the skull is non-zero. Once these thresholds are defined, material densities are assigned by the PCTPS for bone, soft tissue, and air (Figure 4).</li>
	<li>If only MRI guidance is needed, load the MRI scan and co-register with the planning CT using the PCTPS.
	<ol>
		<li>Use rigid body transformations (three translations and three rotations), the multimodality markers, and the skull. By overlaying the increased signal intensity of the skull on CT with black signal on MRI, a precise fusion can be achieved (Figure 5).</li>
		<li>Select the target for irradiation in the center of the contrast-enhancing tumor on T1-weighted MRI, see Figure 6 and Figure 7.</li>
	</ol>
	</li>
	<li>When additional PET information must be included, include a CT/MRI/PET co-registration using the biomedical image quantification software (BIQS).
	<ol>
		<li>Use the contouring tool in the BIQS to achieve PET/MRI image fusion (Figure 8). After co-registration, select the target in the center of the increased PET tracer uptake in the BIQS (Figure 9) and enter the coordinates manually into the PCTPS using the following transformations: X &#8594; -X, Y&#160;&#8594; Z, and Z&#160;&#8594; -Y.</li>
		<li>Select the prescribed dose, number of arcs, arc position, rotation range of the arcs, and the collimator size (Figure 10).</li>
		<li>For MRI-guided RT, use the following settings: a prescribed dose of 20 Gy, 3 arcs positioned at couch angles of -45 &#176;, 0 &#176;, and 45 &#176; with arc rotations of 120 &#176;, and a collimator size of 5 x 5 mm.</li>
		<li>For PET-MRI-guided RT, use the following settings: a prescribed dose of 20 Gy using 3 arcs and a 5 x 5 mm collimator and extra 5 Gy for sub-volume boosting using 3 non-coplanar arcs and a 1 x 1 mm collimator. Select a rotation of 120 &#176; for all arcs while changing the position of the couch (-45 &#176;, 0 &#176;, and 45 &#176;).</li>
	</ol>
	</li>
	<li>Calculate the dose distribution within the animal and the beam delivery parameters to deliver the prescribed dose to the target using the PCTPS. Before actual irradiation, test the arc rotations at the different couch positions to prevent any collision during irradiation.</li>
	<li>For the actual irradiation, select a 0.15 mm copper filter, set the X-ray voltage to 220 kV, set the X-ray current to 13 mA, and position the right collimator on the gantry. Execute the RT by transferring the appropriate beam delivery parameters from the PCTPS to the micro-irradiator.</li>
	<li>During these procedures, the rat is kept under continuous isoflurane anesthesia (2% isoflurane, mixed with oxygen 0.3 L/min). Following the execution of the last arc, the animal can wake up under continuous supervision, until it regains full consciousness.</li>
</ol>
5. Dose Volume Histograms (DVHs)
NOTE: To compare the actual dose delivered to the tumor target volumes and the surrounding normal brain tissue, calculate DVHs.
<ol>
	<li>Draw a volume-of-interest (VOI) around the tumor and the normal brain on the T1-weighted contrast-enhanced MR images to calculate the mean, maximum, and minimum dose (Figure 11).</li>
	<li>As a surrogate for the maximal, mean, and minimal dose to the tumor volume and the normal brain tissue volume, calculate the D2, D50, and D90. D stands for the dose received by x% of the volume, denoted by the subscript, and can be derived from the resulting DVH.</li>
</ol>
6. TMZ and Sham Chemotherapy
<ol>
	<li>To mimic the treatment of glioblastoma in patients, administer concomitant chemotherapy using IP injections of 29 mg/kg TMZ dissolved in saline with 25% dimethylsulfoxide (DMSO) once a day for 5 days starting at the day of irradiation24,25. Use 1 mL, 29 G insulin syringe to administer injection.</li>
	<li>For the control group, administer injection from step 6.1 without TMZ.</li>
</ol>

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The authors have no conflicts of interest to disclose

To achieve accurate irradiation of the glioblastoma tumor target in the rat brain, the micro-irradiator&#39;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...

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 TMZ1,2,3. 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 animal cancer therapy models (mostly mice and rats). Tumor-bearing rodent models can be used to investigate the efficacy of new and complex radiation protocols, possibly combined with other (new) treatment agents, to assess radiation response or to investigate radio-protective agents. A major advantage of preclinical radiation research is the ability to work under controlled experimental conditions using large cohorts resulting in accelerated data yield due to the shorter lifespans of rodents. The preclinical findings should then be translated into a clinical trial in a much faster and more efficient way than in current practice4.
Small animal radiation experiments in the last decades have typically been achieved using fixed radiation sources5,6,7, e.g., 137Cs and 60Co, isotopes, or linear accelerators intended for human clinical use, applying a single radiation field with MV X-rays6,8,9,10,11. However, these devices do not reach sub-millimeter precision, which is required for small animals12. Furthermore, MV X-rays have characteristics unsuitable for irradiating small targets, such as a dose build-up at the air-tissue interface in the entrance region of the beam with an extent in the order of the animal size itself4,6,8,9,10,11. The latter makes it quite challenging to deliver a uniform dose to a tumor while sparing surrounding normal brain tissue4,8,9,10,11. Hence, it is unclear to which extent current animal studies still are relevant for modern RT practice12. In this respect, recently developed three-dimensional (3D) conformal small animal micro-irradiators are promising to bridge the technological gap between advanced 3D image-guided RT techniques, such as intensity modulated radiation therapy (IMRT) or conformal arcs used in humans and current small animal irradiation4,13. These platforms make use of a kilovoltage (kV) X-ray source to obtain sharp penumbras and to avoid dose build-up. These platforms include a computer-controlled stage for animal positioning, a kV X-ray source for imaging and radiation treatment, a rotational gantry assembly to allow radiation delivery from various angles, and a collimating system to shape the radiation beam4. In 2011, a micro-irradiator was installed at the preclinical imaging lab of Ghent University (Figure 1). This system is similar to modern human radiotherapy practice and enables a wide variety of preclinical experiments, such as the synergy of radiation with other therapies, complex radiation schemes, and image-guided sub-target boost studies.
Treatment planning on these micro-irradiators is based on CT, which is equivalent to human planning systems14,15. For CT imaging, an on-board X-ray detector is used in combination with the same kV X-ray tube that is used during treatment. CT imaging is used as it allows for accurate animal positioning and provides information necessary for individual radiation dose calculations via segmentation. However, due to the low soft-tissue contrast in CT imaging, tumors in the brain of small animals, such as high-grade glioma, cannot be easily delineated. The incorporation of multi-modality imaging is therefore necessary for an accurate target volume delineation. Compared to CT, MRI provides vastly superior soft-tissue contrast. This makes it much easier to visualize lesion boundaries that will result in a much better delineation of the target volume, helping to better irradiate the lesion and avoid surrounding tissue, as illustrated in Figure 24,16. An additional advantage is that MRI uses non-ionizing radiation, unlike CT that is using ionizing radiation. The major disadvantages of MRI are the relatively long acquisition times and high operational costs. It is important to note that MRI scans cannot be used for dose calculations, as they do not provide the required electron density information, although progress is being made in this field, too with the recent development of MR-LINACS. As such, a combined CT/MRI dataset is the method of choice for planning the irradiation of malignant glioma, containing both the information required for targeting (MRI-based volumes) and for dose calculations (CT-based electron density).
To decrease the gap between small animal irradiation and clinical routine, MRI clearly needs to be integrated into the work flow of the micro-irradiator, requiring a correct registration between MRI and CT, which is far from trivial. In this paper, our protocol for MRI-guided 3D conformal irradiation of F98 glioblastoma in rats is discussed, which has been published recently17.
Although incorporating CT and MRI in the workflow of the micro-irradiator is a clear step forward in small animal irradiation research, these anatomical imaging techniques do not always allow a full definition of the target volume. Pathological changes in the brain on CT and MRI are characterized by increased water content (edema) and leakage of the blood-brain barrier or contrast enhancement. However, both contrast-enhancement and hyper-intense areas on T2-weighted MRI are not always an accurate measure of tumor extent. Tumor cells have been detected far beyond the margins of contrast-enhancement12. Also, none of these techniques can identify the most aggressive parts within the tumor, which may be responsible for therapeutic resistance and tumor recurrence. Therefore, additional information from molecular imaging techniques like PET may have an added value for RT target volume definition because these techniques enable to visualize biologic pathways in vivo12,18,19.
In 2000, Ling et al. introduced the concept of biological target volume (BTV) by integrating anatomical and functional imaging into the radiotherapy workflow, leading to what they called multidimensional conformal radiotherapy20. This creates the possibility to improve dose targeting by delivering a non-uniform dose to a target region using for example PET images. The most widely used PET tracer for tumor staging and to monitor treatment response is fluor-18 (18F) labeled fluorodeoxyglucose (FDG), which visualizes the glucose metabolism21. In head and neck cancer, previous studies have shown that the use of 18F-FDG PET led to a better estimate of the actual tumor volume, as defined by the pathologic specimens, compared with CT and MRI22. In primary brain tumors, where FDG is not useful due to the very strong background signal from the normal brain, amino acids, such as 11C-methionine and more recently 18F-fluoroetthyltyrosine (FET), have been investigated for GTV delineation with often marked differences between amino-acid PET and MRI-based GTVs23. However, no prospective trial investigating the meaning of this finding has been performed yet. In this study, we selected the amino-acid tracer 18F-FET and the hypoxia tracer 18F-fluoroazomycin-arabinoside (18F-FAZA). 18F-FET and 18F-FAZA were selected because an increased amino-acid uptake is strongly correlated with the proliferation rate in GB tumors, whereas uptake of a hypoxia PET-tracer is correlated with resistance to (chemo)radiotherapy18,23. Sub-volume boosting using the micro-irradiator was optimized by giving an additional radiation dose to a PET-defined part of the F98 GB tumor in rats.

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

pet and mri guided irradiation of a glioblastoma rat model using a micro-irradiator

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.

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...

Watch this Scientific Journal Video about PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator at JoVE.com

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

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.

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without ...

pet-mri-guided-irradiation-glioblastoma-rat-model-using-micro

Research

JoVE Journal

Cancer Research

9.5K Views.  Ghent University Hospital. Kleine dierlijke bestraling werd in het verleden meestal uitgevoerd zonder de mogelijkheid om gericht een goed afgebakende tumor volume. Het doel was om na te bootsen de behandeling van menselijke glioblastoma bij ratten. Met behulp van een kleine dierlijke bestraling platform, wij uitgevoerd MRI geleide 3D hoekgetrouwe bestraling met PET gebaseerde sub volume boost in een preklinische instelling.

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

Stem Cell Transplantation Strategies for the Restoration of Cognitive Dysfunction Caused by Cranial Radiotherapy

Radiotherapy often provides the only clinical recourse for those afflicted with primary or metastatic brain tumors. While beneficial, cranial irradiation can induce a progressive and debilitating decline in cognition that may, in part, be caused by the depletion of neural stem cells. Given the increased survival of patients diagnosed with brain cancer, quality of life in terms of cognitive health has become an increasing concern, especially in the absence of any satisfactory long-term treatments.
To address this serious health concern we have used stem cell replacement as a strategy to combat radiation-induced cognitive decline. Our model utilizes athymic nude rats subjected to cranial irradiation. The ionizing radiation is delivered as either whole brain or as a highly focused beam to the hippocampus via linear accelerator (LINAC) based stereotaxic radiosurgery. Two days following irradiation, human neural stem cells (hNSCs) were stereotaxically transplanted into the hippocampus. Rats were then assessed for changes in cognition, grafted cell survival and for the expression of differentiation-specific markers 1 and 4-months after irradiation. Our cognitive testing paradigms have demonstrated that animals engrafted with hNSCs exhibit significant improvements in cognitive function. Unbiased stereology reveals significant survival (10-40%) of the engrafted cells at 1 and 4-months after transplantation, dependent on the amount and type of cells grafted. Engrafted cells migrate extensively, differentiate along glial and neuronal lineages, and express a range of immature and mature phenotypic markers.
Our data demonstrate direct cognitive benefits derived from engrafted human stem cells, suggesting that this procedure may one day afford a promising strategy for the long-term functional restoration of cognition in individuals subjected to cranial radiotherapy. To promote the dissemination of the critical procedures necessary to replicate and extend our studies, we have provided written and visual documentation of several key steps in our experimental plan, with an emphasis on stereotaxic radiosurgey and transplantation.

Brain tumor patients routinely undergo cranial radiotherapy, and while beneficial, this treatment often results in debilitating cognitive dysfunction. This serious unresolved problem has at present, no clinical recourse, and has driven our efforts to devise stem cell based therapies for the recovery of radiation-induced cognitive decrements.

Radiotherapy often provides the only clinical recourse for those afflicted with primary or metastatic brain tumors. While beneficial, cranial irradiation ...

Medicine

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain 1; therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation 1.
Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4mm to a depth of 2.6mm. Two &mu;l of GL261-luc or GL261-luc2 cells (107 cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured.
Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150&mu;g luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in &gt;10% loss of weight. Tumors were evident in all of the animals on necropsy.

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intracranial implantation of GL261 cells into C57BL/6 mice produces malignant gliomas that recapitulate many of the hallmarks of human glioblastoma multiforme. We used GL261 cells stably expressing luciferase to allow us to use in vivo imaging to follow tumor progression. The surgery and 3D in vivo imaging are demonstrated.

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

Positron Emission Tomography-based Dose Painting Radiation Therapy in a Glioblastoma Rat Model using the Small Animal Radiation Research Platform

A rat glioblastoma model to mimic chemo-radiation treatment of human glioblastoma in the clinic was previously established. Similar to the clinical treatment, computed tomography (CT) and magnetic resonance imaging (MRI) were combined during the treatment-planning process. Positron emission tomography (PET) imaging was subsequently added to implement sub-volume boosting using a micro-irradiation system. However, combining three imaging modalities (CT, MRI, and PET) using a micro-irradiation system proved to be labor-intensive because multimodal imaging, treatment planning, and dose delivery have to be completed sequentially in the preclinical setting. This also results in a workflow that is more prone to human error. Therefore, a user-friendly algorithm to further optimize preclinical multimodal imaging-based radiation treatment planning was implemented. This software tool was used to evaluate the accuracy and efficiency of dose painting radiation therapy with micro-irradiation by using an in silico study design. The new methodology for dose painting radiation therapy is superior to the previously described method in terms of accuracy, time efficiency, and intra- and inter-user variability. It is also an important step towards the implementation of inverse treatment planning on micro-irradiators, where forward planning is still commonly used, in contrast to clinical systems.

Here we present a protocol to perform preclinical positron emission tomography-based radiotherapy in a rat glioblastoma model using algorithms developed in-house to optimize the accuracy and efficiency.

A rat glioblastoma model to mimic chemo-radiation treatment of human glioblastoma in the clinic was previously established. Similar to the clinical ...

Magnetic Resonance-Guided High Intensity Focused Ultrasound Generated Hyperthermia: A Feasible Treatment Method in a Murine Rhabdomyosarcoma Model

Magnetic resonance-guided high intensity focused ultrasound (MRgHIFU) is an established method for producing localized hyperthermia. Given the real-time imaging and acoustic energy modulation, this modality enables precise temperature control within a defined area. Many thermal applications are being explored with this noninvasive, nonionizing technology, such as hyperthermia generation, to release drugs from thermosensitive liposomal carriers. These drugs can include chemotherapies such as doxorubicin, for which targeted release is desired due to the dose-limiting systemic side effects, namely cardiotoxicity. Doxorubicin is a mainstay for treating a variety of malignant tumors and is commonly used in relapsed or recurrent rhabdomyosarcoma (RMS). RMS is the most common solid soft tissue extracranial tumor in children and young adults. Despite aggressive, multimodal therapy, RMS survival rates have remained the same for the past 30 years. To explore a solution for addressing this unmet need, an experimental protocol was developed to evaluate the release of thermosensitive liposomal doxorubicin (TLD) in an immunocompetent, syngeneic RMS mouse model using MRgHIFU as the source of hyperthermia for drug release.

Presented here is a protocol to use controlled hyperthermia, generated by magnetic resonance-guided high intensity focused ultrasound, to trigger drug release from temperature-sensitive liposomes in a rhabdomyosarcoma mouse model.

Magnetic resonance-guided high intensity focused ultrasound (MRgHIFU) is an established method for producing localized hyperthermia. Given the real-time ...

Real-Time Tracking of Nanoparticles in Brain Tumors Using 2P-IVM in Mice

Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the accumulation and distribution of macromolecules in brain tumors in vivo would greatly enhance our ability to understand and optimize drug delivery in preclinical models. This protocol describes a method for real-time in vivo tracking of intravenously administered fluorescent-labeled nanoparticles with two-photon intravital microscopy (2P-IVM) in a mouse model of glioblastoma (GBM).
The protocol contains a multi-step description of the procedure, including anesthesia and analgesia of experimental animals, creating a cranial window, GBM cell implantation, placing a head bar, conducting 2P-IVM studies, and post-surgical care for long-term follow-up studies. We show representative 2P-IVM imaging sessions and image analysis, examine the advantages and disadvantages of this technology, and discuss potential applications.
This method can be easily modified and adapted for different research questions in the field of in vivo preclinical brain imaging.

Author Spotlight: Multimodal Imaging Strategies for Optimizing Drug Delivery and Early Detection in Glioblastoma Treatment

We present a novel approach for two-photon microscopy of the tumor delivery of fluorescent-labeled iron oxide nanoparticles to glioblastoma in a mouse model.

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the ...

Iron Oxide Nanoparticles for Image-Guided Therapy Delivery in Murine Glioblastoma

Nanoparticle Delivery of an Oligonucleotide Payload in a Glioblastoma Multiforme Animal Model

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a significant hurdle in the delivery of therapies to GBM. Reported here is an image-guided, iron oxide-based therapeutic delivery nano platform capable of bypassing this physiological barrier by virtue of size and accumulating in the tumor region, delivering its payload. This 25 nm nano platform consists of crosslinked dextran-coated iron oxide nanoparticles labeled with Cy5.5 fluorescent dye and containing antisense oligonucleotide as a payload. The magnetic iron oxide core enables tracking of the nanoparticles through in vivo magnetic resonance imaging, while Cy5.5 dye allows tracking by optical imaging. This report details the monitoring of the accumulation of this nanoparticle platform (termed MN-anti-miR10b) in orthotopically implanted glioblastoma tumors following intravenous injection. In addition, it provides insight into the in vivo delivery of RNA oligonucleotides, a problem that has hampered the translation of RNA therapeutics into the clinic.

Author Spotlight: Innovative Cancer Therapies with Iron Oxide Nanoparticles for Glioblastoma Treatment

The blood-brain barrier is a significant hurdle in the delivery of therapies for glioblastoma, a disease for which there is no cure. Here, we report an in vivo image-guided iron oxide therapeutic nano platform that can bypass this physiological barrier by virtue of size and accumulate in the tumor.

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a ...

An Immunocompetent Murine Model for Laser Interstitial Thermal Therapy of Glioblastoma

Glioblastoma (GB), the most aggressive form of primary brain cancer, accounts for approximately half of all high-grade primary brain tumors in adults and has no cure. Laser interstitial thermal therapy (LITT) is a Food and Drug Administration (FDA)-approved treatment for GB and is used in patients who may not be candidates for conventional surgical resection. While the clinical efficacy of LITT has been established, research beyond clinical case studies and case series is limited and hindered by the lack of an established animal model. This protocol uses C57BL/6 mice and syngeneic CT2A glioma cancer cell line to closely recapitulate human GB&#160;while also using a 1064 nm Neodymium-doped Yttrium Aluminum Garnet (Nd:YAG) laser, such as is used in one of the two FDA-approved LITT systems, providing excellent pre-clinical relevance. The successful establishment of this LITT murine model will provide a valuable platform for investigating the unique features of LITT ablation and its effects on the tumor microenvironment, potentially leading to improved therapeutic strategies.

Glioblastoma is a devastating form of primary brain cancer, and laser interstitial thermal therapy is emerging as a promising alternative to conventional surgical resection for inoperable glioblastoma. This protocol describes an optimized pre-clinical mouse model that can be used to study treatment effects or adjuvant and combinatorial treatments.

Glioblastoma (GB), the most aggressive form of primary brain cancer, accounts for approximately half of all high-grade primary brain tumors in adults and ...

Targeted and Selective Treatment of Pluripotent Stem Cell-derived Teratomas Using External Beam Radiation in a Small-animal Model

The growing number of victims of &#34;stem cell tourism,&#34; the unregulated transplantation of stem cells worldwide, has raised concerns about the safety of stem cell transplantation. Although the transplantation of differentiated rather than undifferentiated cells is common practice, teratomas can still arise from the presence of residual undifferentiated stem cells at the time of transplant or from spontaneous mutations in differentiated cells. Because stem cell therapies are often delivered into anatomically sensitive sites, even small tumors can be clinically devastating, resulting in blindness, paralysis, cognitive abnormalities, and cardiovascular dysfunction. Surgical access to these sites may also be limited, leaving patients with few therapeutic options. Controlling stem cell misbehavior is, therefore, critical for the clinical translation of stem cell therapy.
External beam radiation offers an effective means of delivering targeted therapy to decrease the teratoma burden while minimizing injury to surrounding organs. Additionally, this method avoids genetic manipulation or viral transduction of stem cells-which are associated with additional clinical safety and efficacy concerns. Here, we describe a protocol to create pluripotent stem cell-derived teratomas in mice and to apply external beam radiation therapy to selectively ablate these tumors in vivo.

Research on treatment strategies for pluripotent stem cell-derived teratomas is important for the clinical translation of stem cell therapy. Here, we describe a protocol to, first, generate stem cell-derived teratomas in mice and, then, to selectively target and treat these tumors in vivo using a small-animal irradiator.

The growing number of victims of &#34;stem cell tourism,&#34; the unregulated transplantation of stem cells worldwide, has raised concerns about the ...

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

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