Therapeutic exposures were performed using 220 kVp X-rays (broad focus, 0.15 mm Cu filter and a half value layer of 0.93 mm Cu) delivered using the SARRP irradiator in conjunction with collimators to produce a range of field sizes. Additional filtration results from the presence of a transmission monitor (in total: 0.105 mm Cu and 1 mm of FR4 a fibreglass/epoxy mix). For each collimator, dosimetry was performed using an EBT3 film as described before, with the film calibrated following the recommendations of the report of the American Association of Physicists in Medicine Task Group 61 [20 (link), 21 (link)]. Treatment planning and subsequent beam delivery was performed using Muriplan (Xstrahl Ltd, Camberley, Surrey, UK), with targeting and subsequent segmentation performed using the CBCT image [22 (link)].
Muriplan
Manufactured by Xstrahl
Sourced in United Kingdom
Muriplan is a lab equipment product designed for the planning and delivery of radiation therapy for small animal research. It provides precise control over radiation dosage and exposure parameters to support scientific investigations.
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Lab products found in correlation
11 protocols using muriplan
1
Small Animal Radiation Research Platform Dosimetry
2
Lung Dosimetry in Mice Post-Irradiation
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CBCT scans were performed prior to irradiation and at 4, 12 and 26 weeks post irradiation.
Lungs were outlined and DVHs were calculated retrospectively for each mouse using Muriplan (Xstrahl Inc., Suwannee, GA). Dosimetric parameters including MLD for the irradiated lung and V10 were extracted from the individual DVHs using Matlab R2017a. The percentage of the irradiated lung receiving at least 50% of the dose was 50.7 % ± 19% for single fraction exposures and 47.2 % ± 24% for fractionated arm of the study. Significant variations in the DVHs were observed as shown in Figure 1b due to anatomical variations for individual mice at the time of the imaging which allowed investigation of dose-volume relationships.
Lungs were outlined and DVHs were calculated retrospectively for each mouse using Muriplan (Xstrahl Inc., Suwannee, GA). Dosimetric parameters including MLD for the irradiated lung and V10 were extracted from the individual DVHs using Matlab R2017a. The percentage of the irradiated lung receiving at least 50% of the dose was 50.7 % ± 19% for single fraction exposures and 47.2 % ± 24% for fractionated arm of the study. Significant variations in the DVHs were observed as shown in Figure 1b due to anatomical variations for individual mice at the time of the imaging which allowed investigation of dose-volume relationships.
3
Targeted Partial Brain Irradiation in Mice
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All in vivo animal experiments were performed in full compliance with the institutional guidelines of the Helmholtz Center Munich and the Technical University of Munich and with approval from the Government District of Upper Bavaria. Image-guided partial irradiation was performed on four-week-old female C57Bl/6 mice (n=7) (Charles River, United States) using a small-animal radiation research platform (SARRP, X-Strahl, United Kingdom). Mice were anesthetized by isoflurane/oxygen inhalation for the duration of the procedure.
Transverse, sagittal and frontal computed tomography scans using 60 kV and 0.8 mA photons were performed for each mouse for precise radiation targeting [26] (link). The irradiation targeted 40% of the left brain volume with a beam size of approximately (6x8) mm² and with a mean target dose of 20 Gy using 220 kV and a 13 mA X-ray beam filtered with copper (0.15 mm). The software SARRP control and Muriplan (both X-Strahl, United Kingdom) were used for CT imaging, precise radiation targeting, and calculation of the irradiation dose. A total of four mice received partial brain irradiation while three mice where sham radiated. All mice fully recovered after the procedure and were housed in single ventilated cages under pathogen-free conditions prior to imaging.
Transverse, sagittal and frontal computed tomography scans using 60 kV and 0.8 mA photons were performed for each mouse for precise radiation targeting [26] (link). The irradiation targeted 40% of the left brain volume with a beam size of approximately (6x8) mm² and with a mean target dose of 20 Gy using 220 kV and a 13 mA X-ray beam filtered with copper (0.15 mm). The software SARRP control and Muriplan (both X-Strahl, United Kingdom) were used for CT imaging, precise radiation targeting, and calculation of the irradiation dose. A total of four mice received partial brain irradiation while three mice where sham radiated. All mice fully recovered after the procedure and were housed in single ventilated cages under pathogen-free conditions prior to imaging.
4
Verifying Treatment Planning System Consistency
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The treatment planning system (TPS) is also checked for consistency. For a scan (same scan data set each time) all parameters like segmentation, isocenter placement, and calculation presets have to be set in the same way each time. Then the needed irradiation time for a certain dose is calculated. In our case we used a 5 × 5 mm2 field size for a fixed beam as well as for an arc (gantry rotation −178° to +178°) to calculate the irradiation time for 10 Gy in the planning system MuriPlan (Xstrahl). In addition, we calculated the time for a static beam with a field size of 40 × 20 mm2 (also 10 Gy) with the variable collimator. We used a dose of 10 Gy to have a treatment time significantly more than 100 s to recognize also small variations. With this check we minimize the risk to have unrecognized changes in the TPS settings including the basic input data and to thereby receive wrong results. A TPS not configured for clinical use (like this one) may be prone to or less protected from such changes (that are sometimes even part of ongoing research projects).
5
Evaluating Anti-VEGFR2 and Anti-Dll4 Antibodies in Tumor Radiotherapy
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Animals with tumors approximately 100 mm3 in the chamber were administered with either anti-mouse vascular endothelial growth factor receptor 2 (VEGFR2) antibody [27 mg/kg; clone DC101 (37 (link)), BioXCell], anti-mouse Dll4 antibody (39 (link)) twice per week at a dose of 5 mg/kg (in two doses on the initial day of imaging and 3 days later), or one of two radiation treatments. For the radiation treatments, mice were anesthetized under inhalation with isoflurane and placed in an imaging-guided small animal radiation research platform (SARRP) irradiator (Xstrahl Ltd). A Cone Beam CT (computerized tomography) scan of each mouse was obtained, and the treatment was planned using MuriPlan (Xstrahl Ltd). The SARRP was used to deliver 15 Gy of x-rays (220–kilovolt peak copper-filtered beam with half-value layer of 0.93 mmCu) to the tumor at 2 Gy/min. This was given either in a single dose or at five daily fractionations of 3 Gy of x-ray radiation to the tumor. Dosimetry of the irradiator was performed as previously described (66 (link)).
6
Preclinical SBRT Irradiation Planning
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The preclinical treatment planning software “MuriPlan” (Xstrahl Ltd., Camberley, UK) with its superposition-convolution algorithm for dose calculation was used for treatment planning. Gross tumor volume (GTV) and six OARs (both kidneys, small bowel, stomach, spinal cord, and liver) were delineated slice by slice on the CBCT image [23 (link)]. The GTV was defined as the macroscopically visible tumor tissue. A safety margin of 2 mm surrounding the GTV was considered. The radiation was delivered at 220 kV and 13 mA with a dose rate of 2.6 Gy/min by the SARRP device.
In the following, the single-dose high-precision irradiation of the GTV technique will be denoted as SBRT. An image-guided single-dose SBRT of 25 Gy compound dose-to-water (to the isocenter, which was in the center of the GTV) was performed in a single-arc technique with a gantry rotation from 178° to −178° with a field size of 10 × 10 mm2 using a fixed collimator. In comparison to the high-precision RT, whole-abdomen irradiation with 25 Gy (normalized to the isocenter, which was in the center of the GTV) was delivered in AP/PA opposing field technique with a gantry angle of 0° and 180° using a variable collimator with a field size of 50 × 30 mm2.
In the following, the single-dose high-precision irradiation of the GTV technique will be denoted as SBRT. An image-guided single-dose SBRT of 25 Gy compound dose-to-water (to the isocenter, which was in the center of the GTV) was performed in a single-arc technique with a gantry rotation from 178° to −178° with a field size of 10 × 10 mm2 using a fixed collimator. In comparison to the high-precision RT, whole-abdomen irradiation with 25 Gy (normalized to the isocenter, which was in the center of the GTV) was delivered in AP/PA opposing field technique with a gantry angle of 0° and 180° using a variable collimator with a field size of 50 × 30 mm2.
7
Precise Image-Guided Tumor Irradiation in Mice
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Mice were anesthetized under inhalation with isoflurane and placed in an imaging‐guided small animal radiation research platform (SARRP) irradiator (Xstrahl Ltd). A cone beam CT image of each mouse was obtained, and the treatment was planned using Muriplan (Xstrahl Ltd) to ensure uniformity of dose across the tumor while sparing the surrounding normal tissue. This was achieved using a coronal arc beam with the isocenter positioned a few millimeters above the glass window with a beam at an angle of 65° to the vertical and the mouse rotated through 360° horizontally. To achieve full coverage of the tumor, a 4 mm × 10 mm field size (defined as the isocenter and the long axis parallel to the mouse) was chosen. The SARRP was used to deliver 15 Gy of X‐rays (220 kVp copper‐filtered beam with HVL of 0.93 mmCu) to the tumor at ~2 Gy per minute; this was given either in a single fraction or five daily fractionations of 3 Gy X‐ray radiation to the tumor. Dosimetry of the irradiator was performed as previously (Hill et al, 2017 (link)). A visualization of the planned dose distribution is presented in Appendix Fig S4 .
8
Irradiation Protocol for Murine Metabolic Studies
9
Targeted Radiation Therapy for Tumor Imaging
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Mice were treated with IG-IR once palpable tumors were detected. Mice were anaesthetized with isoflurane and transferred to the stage of a small animal radiation research platform instrument (SARRP; XStrahl Inc, Sunwanee, GA). Computed tomography (CT) images were acquired and uploaded into the XStrahl MuriPlan treatment planning software. During treatment planning, a heat lamp was placed near the stage, and an operator continually monitored the anesthetized mouse. The CT images plus manual and automatic tissue segmentation were used to adjust the radiation path. The tumor image was contoured from DICOM slices, and that model used to register its size and location and compute the isocenter where the X-ray beams converge. The angles and weighted dose of each beam were adjusted for each mouse to limit radiation exposure to non-tumor tissue, especially bone. Each isocenter received 10 Gy, split between beams, with each beam delivering 20–50% of the total dose. MuriPlan software was used to ensure that the planned treatment would deliver ≥90% of a 10 Gy dose to ≥80% of the tumor. The mice were returned to their cages and subsequently transported to the University of Alberta for virus treatment.
10
Precision Radiation Therapy for Small Animal Models
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