The authors would like to thank Lux Luka Foundation for supporting this work.

The study was approved by the local ethics committee for animal experiments (ECD 18/21).&#160;Anesthesia monitoring is performed by acquiring the respiratory rate of the animals using a sensor.
1. F98 GB rat cell model
<ol>
	<li>Culture the F98 GB cells in a monolayer using Dulbecco&#39;s Modified Eagle Medium, supplemented with 10% calf serum, 1% penicillin, 1% streptomycin, and 1% L-glutamine, and place them in a CO2 incubator (5% CO2 and 37 &#176;C).</li>
	<li>Inoculate the glioma cells into the brain of female Fischer F344 rats (body weight 170 g). 
	NOTE: Use sterile instruments and wear sterile gloves at all times.
	<ol>
		<li>Anesthetize the rats through the inhalation of isoflurane (5% induction, 2% maintenance) mixed with oxygen (0.3 mL/min) through a nose cone. Confirm the anesthetization by the absence of withdrawal reflex of the limb, and immobilize the rats in a stereotactic device using fixation points for the nose and ears. Apply a carbomer eye gel to prevent dry eyes under anesthesia. Maintain the body temperature by a thermoregulated heating pad and rectal probe at 37 &#176;C.</li>
		<li>Shave the rat from the eye level to the back of the skull, and disinfect the skin with isobetadine. Inject xylocaine (with adrenaline 1:200000, 0.1 mL) subcutaneously for local anesthesia.</li>
		<li>Expose the skull through a midline scalp incision and make a small hole with a drill tool 3 mm posterior and 3 mm lateral to the bregma in the right hemisphere.</li>
		<li>Insert a stereotactically guided insulin needle (29 G) and inject 5 &#181;L of cell suspension (20,000 F98 GB cells) 3 mm deep using a microsyringe pump controller. Leave the needle in place for 5 min, giving the cell suspension time to diffuse into the tissue.</li>
		<li>Withdraw the syringe slowly and close the hole in the skull with bone wax. Suture the skin (polyamide 6, thickness 4-0) and inject meloxicam subcutaneously (1 mg/kg, 2 mg/mL). Apply xylocaine gel.</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. Do not return the animal to the company of other animals until fully recovered. House all animals under environmentally controlled conditions (12 h light/dark cycle, 20-24 &#176;C, and 40-70% relative humidity) with food and water ad libitum.</li>
		<li>Be sure to monitor the animals daily and maintain a daily health status log by checking their body weight, food, water intake, and their activity and 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
<ol>
	<li>Evaluate tumor growth 8 days post-inoculation. Anesthetize the rats through the inhalation of isoflurane (5% induction, 2% maintenance) mixed with oxygen (0.3 mL/min) through a nose cone. Confirm the anesthetization by the absence of withdrawal reflex of the limb.</li>
	<li>Inject a gadolinium-containing contrast agent (0.4 mL/kg) through an intravenously placed tubing in the lateral tail vein. Cover the animal with a&#160;warm water circulating heating blanket and place them in the MRI bed. Apply a carbomer eye gel to prevent dry eyes under anesthesia. Place the MRI bed in the holder with a Tx/Rx Rat brain volume coil.</li>
	<li>Perform a localizer scan followed by a T2-weighted spin-echo scan to assess tumor growth. Use these T2-MRI sequence settings: repetition time (TR)/echo time (TE) 3661/37.1 ms, 109 &#181;m isotropic in-plane resolution, slice thickness 600 &#181;m, 4 averages, 30 slices, total acquisition time (TA) 9 min 45 s.</li>
	<li>If a tumor is confirmed on the T2-weighted acquisition, perform a T1-weighted contrast-enhanced spin echo scan. Use these T1-MRI sequence settings: TR/TE 1539/9.7 ms, 0.117 mm isotropic in-plane resolution, slice thickness 600 &#181;m, 3 averages, 30 slices, TA 4 min 15 s.</li>
	<li>After the MRI, continuously supervise the animal until it regains full consciousness.</li>
	<li>When the tumor reaches a diameter of 7 to 8 mm, usually observed 12 days after inoculation, select the animal for therapy.</li>
</ol>
3. Multimodality imaging of target volume selection
NOTE: PET/MRI-guided irradiation requires the sequential acquisition of a multimodal dataset. After intravenous administration of the radiotracer, PET imaging is started, followed by contrast-enhanced T1-weighted MRI and finally a treatment-planning CT.
<ol>
	<li>Anesthetize the animal with isoflurane (5% induction, 2% maintenance) mixed with oxygen (0.3 L/min) using a nose cone. Confirm anesthetization when the rats do not exhibit any withdrawal reflex of the limb. Apply carbomer eye gel to prevent dry eyes under anesthesia.</li>
	<li>Insert the tubing intravenously in the lateral tail vein, enabling the injection of 10-12 MBq of PET radioactive tracer dissolved in 200 &#181;L of saline. Inject [18F]-FET, 1 h before PET acquisition. Let the animal regain consciousness while the tracer is distributed through the body.</li>
	<li>Anesthetize the animal again, as described in step 3.1. Place the animal on a multimodality bed (here, made in-house) and secure it using hook-and-loop fasteners, maintaining a fixed position during the imaging and micro-irradiation. Fix a capillary filled with the MRI/PET agent (see the Table of Materials) for easier co-registration. Wrap the animal in bubble wrap to preserve its body temperature during the multimodality imaging and therapy.</li>
	<li>Perform a PET scan 1 h after the injection of the PET tracer. Reconstruct the PET scan into a 3D volume (192 x 192 x 384 matrix) with 0.4 mm voxel size by applying a Maximum-Likelihood Expectation-Maximization (MLEM)-algorithm using 30 iterations. 
	NOTE: A dedicated PET scanner for laboratory animal imaging was used with an axial field of view of 130 mm and a bore diameter of 72 mm. This system provides sub-mm (0.85 mm) spatial resolution.</li>
	<li>Inject an MRI contrast agent (0.4 mL/kg) intravenously into the tail vein. Place the rat, still fixed on the multimodality bed, in the animal holder of the MRI scanner (Table of Materials). Perform a localizer scan followed by a contrast-enhanced T1-weighted spin-echo sequence, analogous to step 2.4.</li>
	<li>Place the animal, still fixed on the multimodality bed, on a plastic holder secured onto the four-axis robotic positioning table on the micro-irradiator. Perform a high-resolution treatment-planning cone-beam CT using a tube voltage of 70 kV, a tube current of 0.4 mA, a 1 mm aluminum filter, and a 20 x 20 cm (1024 x 1024 pixel) amorphous Si flat panel detector. Acquire a total of 360 projections over 360&#176;. Reconstruct the CT images with an isotropic voxel size of 0.275 mm (411 x 411 x 251 matrix).</li>
</ol>
4. Image co-registration
NOTE: The co-registration is performed with a semi-automatic MATLAB code developed in-house. The code can be found on Github at https://github.com/sdonche/DosePainting.&#160;The different steps are described below.
<ol>
	<li>Place the three image modalities ([18F]FET PET, contrast-enhanced T1-weighted MRI, and cone-beam CT) into one folder. Convert DICOM images to the NIfTI format using the dcm2niix function from the mricron image viewer24.</li>
	<li>Import the converted images into MATLAB and filter the PET image with a Gaussian filter using a Full-Width Half-Max (FWHM) of 1 mm.</li>
	<li>Reorient the images so that the cartesian axes from all imaging modalities correspond with each other. 
	NOTE: For this setup, the CT image was flipped around the Y-axis; the MRI was flipped around the X-axis, and the PET was flipped around the Y-axis.</li>
	<li>Crop the PET image to simplify automatic co-registration. 
	NOTE: For this setup, 40 pixels were set to zero from both sides of the X-axis (left and right of the animal); on the dorsal and ventral side of the animal (Y-axis), 60 and 40 pixels were set to zero, respectively; along the longitudinal axis (or Z-axis), 170 and 30 pixels are set to zero for inferior and superior side, respectively.</li>
	<li>Move the image centers close to each other to simplify automatic co-registration.</li>
	<li>Perform the actual rigid-body co-registration using Statistical Parametric Mapping (SPM) in MATLAB26. Use the following registration parameters (others on default): objective function: mutual information; separation: [4 1 0.2]; tolerances: [0.02 0.02 0.02 0.001 0.001 0.001 0.01 0.01 0.01 0.001 0.001 0.001]; histogram smoothing: [1 1]; interpolation: trilinear.</li>
</ol>
5. Radiation treatment planning
NOTE: A MATLAB app and multiple MATLAB scripts were written for the radiation treatment planning. The code can be found on Github at https://github.com/sdonche/DosePainting.&#160;The different steps are explained below.
<ol>
	<li>Method 1
	<ol>
		<li>Load the three different imaging modalities into the MATLAB app. Place a generous bounding box around the contrast enhancement on the T1-weighted MRI scan (Figure 1). Determine the contrast-enhanced volume using a threshold (Figure 2). If multiple regions have been selected, select only the largest volume, the center of which is considered as the first isocenter to deliver a prescribed dose for RT (Figure 3).</li>
		<li>Expand the previously determined MRI contrast enhancement by 10 pixels in each direction. If multiple regions are detected, retain only the largest PET volume, the center of which is considered the second isocenter to deliver a prescribed dose for RT. 
		NOTE: In this PET volume, the PET boost volume is defined by the pixels with a higher signal intensity than 0.90 &#215; maximal signal intensity (in the bounding box) in this volume.</li>
		<li>Use the following irradiation settings for the calculated isocenters (Figure 4 and Table 1).
		<ol>
			<li>For the first isocenter (MRI), give a prescribed dose of 2000 cGy using 3 non-coplanar arcs at couch positions 0&#176;, -45&#176;, and -90&#176; with a gantry rotation of 120&#176;, 120&#176;, and 60&#176;, respectively. Use a fixed collimator size of 10 x 10 mm, but use an appropriate collimator (e.g., a 5 x 5 mm collimator) when smaller tumor sizes need to be irradiated. Be careful in considering the animal&#39;s welfare when the tumor volumes are larger than 10 mm.</li>
			<li>For the second isocenter (PET), give a prescribed dose of 800 cGy using 3 non-coplanar arcs at couch positions 0&#176;, -45&#176;, and -90&#176; with a gantry rotation of 120&#176;, 120&#176;, and 60&#176;, respectively. Use a fixed collimator size of 3 x 3 mm.</li>
		</ol>
		</li>
		<li>Calculate the dose distribution within the animal and the beam delivery parameters.</li>
	</ol>
	</li>
	<li>Method 2
	<ol>
		<li>Load the three different imaging modalities into the MATLAB app. Place a generous bounding box around the contrast-enhancement on the [18F]FET PET image, analogous to step 5.1.1.</li>
		<li>Determine the volumes defined by the pixels with a signal intensity higher than A &#215; maximal signal intensity (in the aforementioned bounding box), with A equal to 0.50, 0.60, 0.70, 0.80, and 0.90. Name these volumes V50, V60, V70, V80, and V90, respectively.</li>
		<li>Determine the isocenters and the jaw dimensions for each beam required to guide the motorized variable collimator using the MATLAB script (see Figure 5).</li>
		<li>Use the following settings for the calculated isocenters and jaw dimensions:
		<ol>
			<li>For V50, give a prescribed dose of 2000 cGy distributed over 16 beams (each 125 cGy; couch and gantry positions in Table 2). Use the calculated jaw dimensions for the MVC. 
			NOTE: Here, an additional margin of 1 mm has been included to account for microscopic tumor infiltration.</li>
			<li>For V60-V90, give a prescribed dose of 800 cGy distributed over 40 beams (each 20 cGy; couch and gantry positions in Table 2). Use the calculated jaw dimensions for the MVC.</li>
		</ol>
		</li>
		<li>Calculate the dose distribution within the animal and the beam delivery parameters.</li>
	</ol>
	</li>
</ol>
6. Plan evaluation
NOTE: To compare the two methods, calculate the dose-volume histograms (DVH) and Q-volume histogram (QVH) in the V50 PET volume. Here, a MATLAB script, developed in-house, was used. The code can be found on Github at https://github.com/sdonche/DosePainting.
<ol>
	<li>Dose-volume histogram
	<ol>
		<li>Generate DVH from the dose distribution that was obtained from the SARRP.</li>
		<li>Determine the maximum, mean, and minimum doses from the DVH by calculating the D10, D50, and D90, where Dx stands for the dose received by x% of the volume.</li>
	</ol>
	</li>
	<li>Q-volume histogram
	<ol>
		<li>Calculate an ideal dose for every pixel using Eq. 1, which is a linear interpolation between the minimum and maximum doses, proportionally varying between the minimum PET intensity and maximum PET intensity within the target volume to give an ideal dose map.</li>
		<li>Calculate the Q-value Qp for every pixel using the following equation (Eq. 2): 
		<img alt="Equation 2" src="/files/ftp_upload/62560/62560eq02.jpg" />&#160;Eq. 2 
		With Dp being the dose obtained by planning and Di, the dose objective for planning.</li>
		<li>Generate QVH from the obtained Q-values.</li>
		<li>Calculate the quality factor (Q-factor, QF) to evaluate the difference between the planned and intended doses using Eq. 3: 
		<img alt="Equation 3" src="/files/ftp_upload/62560/62560eq03.jpg" />&#160;Eq. 3</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts of interest to disclose.

A rat GB model to mimic the chemo-radiation treatment in the clinic for glioblastoma patients was previously described20. Similar to the clinical method, CT and MRI were combined during the treatment-planning process to obtain more precise irradiation. A multimodality bed to minimize (head) movement was used when the animal was moved from one imaging system to another. Subsequently, PET imaging was added to the treatment-planning process, and PET-based sub-volume boosting could be successfully imp...

Glioblastoma (GB) is a malignant and very aggressive primary brain tumor. GB is a solid heterogeneous tumor typically characterized by infiltrative boundaries, nuclear atypia, and necrosis1. The presence of the blood-brain-barrier and the brain&#39;s status as an immune-privileged site makes the discovery of novel targets for chemo- and immunotherapy a daunting task2,3,4. It is noteworthy that the treatment of GB patients has barely changed since the introduction, in 2005, of the Stupp protocol that combines external beam radiation therapy (RT) with concomitant temozolomide, usually followed by adjuvant temozolomide5. Typically, the Stupp protocol is preceded by maximal surgical resection. Therefore, alternative treatment approaches are of pivotal importance.
Current radiation therapy for glioblastoma patients delivers a uniform radiation dose to the defined tumor volume. In radiation oncology, there is an important dose-response correlation for glioblastoma with increasing dose, which seems to cap around 60 Gy, due to increased toxicity to the normal brain6,7. However, tumors can be very (radiobiologically) heterogeneous, with gradients of oxygen level and/or large differences in cellular density. Metabolic imaging techniques, such as PET, can visualize these biological features and can be utilized to customize the dose prescription. This approach is known as dose painting RT. This term was introduced by Ling et al. in 2000. The authors defined dose painting RT as producing &#34;exquisitely conformal dose distributions within the constraints of radiation propagation and scatter&#34;8.
There are two types of dose painting RT, dose painting by contours (DPBC), by which a dose is prescribed to a set of nested sub-volumes, and dose painting by numbers (DPBN), whereby a dose is prescribed at the voxel level. The dose distribution for DPBN RT can be extracted from functional images. The dose in each voxel is determined by the intensity I of the corresponding voxel in the image, with a lower and upper limit, to make sure that, on the one hand, a sufficient dose is delivered to every part of the tumor. On the other hand, doses&#160;do not exceed an upper limit to protect organs at risk and avoid toxicity. The most straightforward method is a linear interpolation (see Eq. 1) between minimum dose&#160;Dmin and maximum dose Dmax, proportionally varying between minimum intensity Imax and maximum intensity within the target volume9,10
<img alt="Equation 1" src="/files/ftp_upload/62560/62560eq01.jpg" />&#160;Eq. 1
Because there is some skepticism about the quality assurance of DPBN RT, the deposition of the dose should be verified through preclinical and clinical research10. However, only limited data can be acquired from clinical trials, and it has been hypothesized that more insights can be obtained by downscaling to laboratory animals11,12. Hence, preclinical studies utilizing precision image-guided radiation research platforms that allow coupling with some very specific techniques, such as autoradiography, are suited for examining open issues and paving the way towards personalized medicine and novel treatment strategies, such as dose painting RT13,14. However, the interpretation of preclinical data must be performed with caution, and drawbacks of these preclinical setups have to be considered14.
Micro-irradiation systems, such as the Small Animal Radiation Research Platform (SARRP), are equipped with similar technologies as their clinical counterpart. They include on-board cone-beam CT (CBCT) imaging, a preclinical treatment-planning system (PCTPS), and provide sub-millimeter precision. Clinical dose calculations are performed by inverse treatment planning, whereby one initiates from the desired dose distribution to determine the beams via an iterative algorithm. Preclinical irradiators often use forward planning. In forward planning, the required amount and angle of the beams are selected, and the PCTPS then calculates the dose distribution. The optimization of the plans is performed by manual iteration, which is labor-intensive15.
After 2009, novel developments have made the implementation of inverse planning on these research platforms possible16,17,18. To increase the similarity with the clinical method, a motorized variable rectangular collimator (MVC) was developed as a preclinical counterpart of the multi-leaf collimator. A two-dimensional dose painting method utilizing a variable collimator was published by Cho et al.19. This research group implemented a three-dimensional (3D) inverse treatment-planning protocol on a micro-irradiator and determined minimum and maximum doses for the target volume and a maximum dose for the organs at risk. These techniques have mainly been evaluated in silico, and their preclinical applications need to be explored.
This paper presents an in silico study to compare two methodologies for [18F]-fluoro-ethyl-L-tyrosine ([18F]FET) PET-based dose painting in a GB rat model20,21,22 using a small animal radiation research platform. These two methodologies are (1) sub-volume boosting using predefined beam sizes and (2) dose painting using a motorized variable collimator where jaw dimensions are modified based on the PET tracer uptake in the tumor volume. [18F]FET is a PET tracer often used in neuro-oncology because of its ability to detect brain tumors23. [18F]FET is an artificial amino acid that is internalized into tumoral cells but not incorporated into cell proteins. [18F]FET uptake corresponds with cell proliferation rate, tumor cell density, and angiogenesis24. As this is the most commonly used oncologic brain PET tracer in these authors&#39; institute, this radiotracer was chosen to evaluate the new workflow.

<table><tbody><tr><td>Cell culture</td><td></td><td></td><td></td></tr><tr><td>F98 Glioblastoma Cell Line</td><td>ATCC</td><td>CRL-2397</td><td>https://www.lgcstandards-atcc.org/products/all/CRL-2397</td></tr><tr><td>Dulbeco&#039;s Modified Eagle Medium</td><td>Thermo Fisher Scientific</td><td>22320-030</td><td></td></tr><tr><td>Cell culture flasks</td><td>Thermo Fisher Scientific</td><td>178883</td><td>75 cm&amp;sup2;</td></tr><tr><td>FBS</td><td>Thermo Fisher Scientific</td><td>10270106</td><td></td></tr><tr><td>L-Glutamine</td><td>Thermo Fisher Scientific</td><td>25030-032</td><td>200 mM</td></tr><tr><td>Penicilline-Streptomycin</td><td>Thermo Fisher Scientific</td><td>15140-148</td><td>10,000 U/mL</td></tr><tr><td>Phosphate-Buffered Saline (PBS)</td><td>Thermo Fisher Scientific</td><td>14040-224</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Thermo Fisher Scientific</td><td>25300-062</td><td>0.05%</td></tr><tr><td>GB Rat Model</td><td></td><td></td><td></td></tr><tr><td>Ball-shaped burr</td><td>Foredom</td><td>A-228</td><td>1.8 mm</td></tr><tr><td>Bone Wax</td><td>Aesculap</td><td>1029754</td><td>https://www.aesculapusa.com/en/healthcare-professionals/or-solutions/or-solutions-cranial-closure/hemostatic-bone-wax.html</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>Fischer F344/Ico crl Rats</td><td>Charles River</td><td>-</td><td></td></tr><tr><td>Insulin Syringe Microfine</td><td>Beckton-Dickinson</td><td>320924</td><td>1 mL, 29 G</td></tr><tr><td>IR Lamp</td><td>Philips</td><td>HP3616/01</td><td></td></tr><tr><td>Meloxicam (Metacam)</td><td>Boehringer Ingelheim</td><td>-</td><td>2 mg/mL</td></tr><tr><td>Micromotor rotary tool</td><td>Foredom</td><td>K.1090-22</td><td></td></tr><tr><td>Micropump system</td><td>Stoelting Co.</td><td>53312</td><td>Stoelting Stereotaxic Injector</td></tr><tr><td>Stereotactic frame</td><td>Stoelting Co.</td><td>51600</td><td></td></tr><tr><td>Xylocaine (1%, with adrenaline 1:200,000)</td><td>Aspen</td><td>-</td><td>1%, with adrenaline 1:200,000</td></tr><tr><td>Xylocaine gel (2%)</td><td>Aspen</td><td>-</td><td>2%</td></tr><tr><td>Animal Irradiation</td><td></td><td></td><td></td></tr><tr><td>Micro-irradiator</td><td>X-Strahl</td><td>SARRP</td><td>Version 4.2.0</td></tr><tr><td>Software</td><td>X-Strahl</td><td>Muriplan</td><td>Preclinical treatment planning system (PCTPC), version 2.2.2</td></tr><tr><td>Small Animal PET</td><td></td><td></td><td></td></tr><tr><td>[&lt;sup&gt;18&lt;/sup&gt;F]FET</td><td>Inhouse made</td><td>-</td><td>PET tracer; along with Prohance: MRI/PET agent</td></tr><tr><td>Micro-PET</td><td>Molecubes</td><td>Beta-Cube</td><td>https://www.molecubes.com/b-cube/</td></tr><tr><td>Small Animal MRI</td><td></td><td></td><td></td></tr><tr><td>Micro-MRI</td><td>Bruker Biospin</td><td>Pharmascan 70/16</td><td>https://www.bruker.com/products/mr/preclinical-mri/pharmascan.html</td></tr><tr><td>30 G Needle for IV injection</td><td>Beckton-Dickinson</td><td>305128</td><td></td></tr><tr><td>PE 10 Tubing</td><td>Instech Laboratories Inc</td><td>BTPE-10</td><td>BTPE-10, polyethylene tubing 0.011 x 0.024 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>Prohance contrast agent</td><td>Bracco Imaging</td><td>-</td><td>279.3 mg/mL, gadolinium-contrast agent (along with [&lt;sup&gt;18&lt;/sup&gt;F]FET: MRI/PET agent)</td></tr><tr><td>Tx/Rx Rat Brain - Mouse Whole Body Volumecoil</td><td>Bruker Biospin</td><td>-</td><td>40 mm diameter</td></tr><tr><td>Water-based Heating Unit</td><td>Bruker Biospin</td><td>MT0125</td><td></td></tr><tr><td>Consumables</td><td></td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Zoetis</td><td>B506</td><td>Anesthesia</td></tr><tr><td>Insulin Syringe Microfine</td><td>Beckton-Dickinson</td><td>320924</td><td>1 mL, 29 G</td></tr><tr><td>Image Analysis</td><td></td><td></td><td></td></tr><tr><td>MATLAB</td><td>Mathworks</td><td>-</td><td>Version R2019b</td></tr><tr><td>PMOD</td><td>PMOD technologies LLC</td><td></td><td>Preclinical and molecular imaging software</td></tr></tbody></table>

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.

The feasibility of PET- and MRI-guided irradiation in a glioblastoma rat model using the SARRP to mimic the human treatment strategy has been previously described20,21,22. While the animal was fixed on a multimodality bed made in-house, it was possible to create an acceptable radiation treatment plan combining three imaging modalities: PET, MRI, and CT. In these methods, an external software package (see the Table of Mat...

Watch this Scientific Journal Video about Positron Emission Tomography-based Dose Painting Radiation Therapy in a Glioblastoma Rat Model using the Small Animal Radiation Research Platform at JoVE.com

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

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 ...

positron-emission-tomography-based-dose-painting-radiation-therapy

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Cancer Research

2.7K Views.  Ghent University. 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.

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

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.

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 ...

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 ...

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 ...

Translational Orthotopic Models of Glioblastoma Multiforme

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&#160;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.

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.

Genetically engineered mouse (GEM) models for human glioblastoma multiforme (GBM) are critical to understanding the development and progression of brain ...

Radiosynthesis and PET/CT Imaging of 68Ga-Labelled Nanobody in Xenograft Models

Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of 68Ga-Labelled Nano Molecules

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical cancer research, necessitating meticulous attention to radiotracer synthesis, quality assurance, and in vivo injection protocols. This study presents a comprehensive workflow tailored to enhance the robustness and reproducibility of small animal PET experiments. The synthesis process in the radiochemistry laboratory using 68Ga is detailed, highlighting stringent quality control and assurance protocols for each radiotracer production. Parameters such as concentration, molar activity, pH, and purity are rigorously monitored, aligning with standards applicable to human studies. This methodology introduces streamlined syringe preparation and a custom-designed 30G cannula for precise intravenous injections into mice. Monitoring of animal health during scanning, including temperature and heart rate, ensures their well-being throughout the procedure. Dosages for PET and SPECT scans are predetermined to balance data acquisition with minimizing radiation exposure to animals and researchers. Similarly, CT scans employ pre-programmed settings to limit radiation exposure, especially pertinent in long-term studies assessing treatment effects. By optimizing these steps, the workflow aims to standardize procedures, reduce variability, and enhance the quality of small animal PET/SPECT/CT imaging. This resource provides valuable insights for researchers seeking to improve the accuracy and reliability of preclinical investigations in molecular imaging, ultimately advancing the field.

This paper describes the radiosynthesis, formulation, quality control of a new radiolabeled probe (i.e., 68Ga-labeled nanobody NM-02), and its use for small animal PET/CT imaging in a xenograft model.

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical ...

Dynamic Lung Tumor Tracking for Stereotactic Ablative Body Radiation Therapy

Physicians considering stereotactic ablative body radiation therapy (SBRT) for the treatment of extracranial cancer targets must be aware of the sizeable risks for normal tissue injury and the hazards of physical tumor miss. A first-of-its-kind SBRT platform achieves high-precision ablative radiation treatment through a combination of versatile real-time imaging solutions and sophisticated tumor tracking capabilities. It uses dual-diagnostic kV x-ray units for stereoscopic open-loop feedback of cancer target intrafraction movement occurring as a consequence of respiratory motions and heartbeat. Image-guided feedback drives a gimbaled radiation accelerator (maximum 15 x 15 cm field size) capable of real-time &#177;4 cm pan-and-tilt action. Robot-driven &#177;60&#176; pivots of an integrated &#177;185&#176; rotational gantry allow for coplanar and non-coplanar accelerator beam set-up angles, ultimately permitting unique treatment degrees of freedom. State-of-the-art software aids real-time six dimensional positioning, ensuring irradiation of cancer targets with sub-millimeter accuracy (0.4 mm at isocenter). Use of these features enables treating physicians to steer radiation dose to cancer tumor targets while simultaneously reducing radiation dose to normal tissues. By adding respiration correlated computed tomography (CT) and 2-[18F] fluoro-2-deoxy-&#7429;-glucose (18F-FDG) positron emission tomography (PET) images into the planning system for enhanced tumor target contouring, the likelihood of physical tumor miss becomes substantially less1. In this article, we describe new radiation plans for the treatment of moving lung tumors.

Stereotactic ablative body radiation therapy (SBRT) involves the precise delivery of high-dose radiation to cancer tumor targets. A novel SBRT platform offers a first-of-its-kind gimbaled radiation accelerator mounted within a pivoting O-ring gantry capable of dynamic image-guided tumor tracking. Here, we describe dynamic tumor tracking for lung targets.

Physicians considering stereotactic ablative body radiation therapy (SBRT) for the treatment of extracranial cancer targets must be aware of the sizeable ...

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 ...

Proton Therapy Delivery and Its Clinical Application in Select Solid Tumor Malignancies

Radiation therapy is a frequently used modality for the treatment of solid cancers. Although the mechanisms of cell kill are similar for all forms of radiation, the in vivo properties of photon and proton beams differ greatly and maybe exploited to optimize clinical outcomes. In particular, proton particles lose energy in a predictable manner as they pass through the body. This property is used clinically to control the depth at which the proton beam is terminated, and to limit radiation dose beyond the target region. This strategy can allow for substantial reductions in radiation dose to normal tissues located just beyond a tumor target. However, the degradation of proton energy in the body remains highly sensitive to tissue density. As a consequence, any changes in tissue density during the course of treatment may significantly alter proton dosimetry. Such changes may occur through alterations in body weight, respiration, or bowel filling/gas, and may result in unfavorable dose deposition. In this manuscript, we provide a detailed method for the delivery of proton therapy using both passive scatter and pencil beam scanning techniques for prostate cancer. Although the described procedure directly pertains to prostate cancer patients, the method may be adapted and applied for the treatment of virtually all solid tumors. Our aim is to equip readers with a better understanding of proton therapy delivery and outcomes in order to facilitate the appropriate integration of this modality during cancer therapy.

The fundamentals of radiation planning and delivery for proton therapy using prostate cancer as a model are presented. The application of these principles to other selected disease sites highlights how proton radiotherapy may enhance clinical outcomes for cancer patients.

Radiation therapy is a frequently used modality for the treatment of solid cancers. Although the mechanisms of cell kill are similar for all forms of ...

Positron Emission Tomography Imaging for In Vivo Measuring of Myelin Content in the Lysolecithin Rat Model of Multiple Sclerosis

Multiple sclerosis (MS) is a neuroinflammatory disease with expanding axonal and neuronal degeneration and demyelination in the central nervous system, leading to motor dysfunctions, psychical disability, and cognitive impairment during MS progression. Positron emission tomography (PET) is an imaging technique able to quantify in vivo cellular and molecular alterations.
Radiotracers with affinity to intact myelin can be used for in vivo imaging of myelin content changes over time. It is possible to detect either an increase or decrease in myelin content, what means this imaging technique can detect demyelination and remyelination processes of the central nervous system. In this protocol we demonstrate how to use PET imaging to detect myelin changes in the lysolecithin rat model, which is a model of focal demyelination lesion (induced by stereotactic injection) (i.e., a model of multiple sclerosis disease). 11C-PIB PET imaging was performed at baseline, and 1 week and 4 weeks after stereotaxic injection of lysolecithin 1% in the right striatum (4 &#181;L) and corpus callosum (3 &#181;L) of the rat brain, allowing quantification of focal demyelination (injection site after 1 week) and the remyelination process (injection site at 4 weeks).
Myelin PET imaging is an interesting tool for monitoring in vivo changes in myelin content which could be useful for monitoring demyelinating disease progression and therapeutic response.

Positron Emission Tomography Imaging for In Vivo Measuring of Myelin Content in the Lysolecithin Rat Model of Multiple Sclerosis

This protocol has the aim of monitoring in vivo myelin changes (demyelination and remyelination) by positron emission tomography (PET) imaging in an animal model of multiple sclerosis.

Multiple sclerosis (MS) is a neuroinflammatory disease with expanding axonal and neuronal degeneration and demyelination in the central nervous system, ...

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

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Chapters in this 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

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

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

Translational Orthotopic Models of Glioblastoma Multiforme

Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of ⁶⁸Ga-Labelled Nano Molecules

Dynamic Lung Tumor Tracking for Stereotactic Ablative Body Radiation Therapy

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

Proton Therapy Delivery and Its Clinical Application in Select Solid Tumor Malignancies

Positron Emission Tomography Imaging for In Vivo Measuring of Myelin Content in the Lysolecithin Rat Model of Multiple Sclerosis