All RT (in vitro and in vivo) was delivered in a single fraction. Delivery of radiation in vitro was performed using a 137Cs-irradiator (JL Shepherd & Associates Model 109) or an X-ray biological cabinet irradiator X-RAD 320 (Precision X-Ray, Inc). Delivery of RT in vivo was performed using an X-ray biological cabinet irradiator X-RAD 320 (Precision X-Ray, Inc). The dose rate for RT delivery in all experiments was approximately 2Gy/min. Dosimetric calibration and monthly quality assurance checks were performed on these irradiators by University of Wisconsin Medical Physics Staff. Unless otherwise indicated, the dose of RT delivered to B78 melanoma cells and tumors was 12 Gy and the dose delivered to A375 melanoma cells was 9 Gy. For B78 melanoma, selection of RT dose was based on our prior findings of a time-sensitive in vivo synergy between single fraction 12 Gy and intra-tumor injection of specific immunotherapies [11 (link)]; that observation provided initial rationale for this study. For A375, which exhibits a greater intrinsic sensitivity to RT, a comparable biologically effective dose (9 Gy) was chosen, as determined by clonogenic survival assays.
X rad 320
Manufactured by Precision X-Ray
Sourced in United States, Canada
The X-RAD 320 is a radiation therapy device that generates X-rays for medical imaging and treatment purposes. It is capable of producing high-energy X-rays for a variety of applications, including diagnostic imaging, image-guided radiation therapy, and small animal research.
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Lab products found in correlation
Orthoclone okt3, by Thermo Fisher Scientific (6 mentions)
Trimethoprim sulfa, by Inotiv (4 mentions)
Gammacell 40 exactor, by Best Theratronics (3 mentions)
Propidium iodide, by Thermo Fisher Scientific (3 mentions)
B6 sjl ptprca pepcb boyj, by Jackson ImmunoResearch (2 mentions)
Cell dissociation medium, by Merck Group (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Optimax, by Precision X-Ray (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Radioflex 200egm, by Rigaku (2 mentions)
Ne2571 farmer ionization chamber, by Thermo Fisher Scientific (2 mentions)
Rs225 cell irradiator, by Xstrahl (2 mentions)
U87 cell line, by BNCC (2 mentions)
Facsaria 2, by BD (2 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Fulvestrant, by Merck Group (1 mentions)
4 hydroxytamoxifen tam, by Merck Group (1 mentions)
Kolliphor el, by Merck Group (1 mentions)
234 protocols using x rad 320
1
Radiation Therapy Delivery Protocol
2
Radiation-Induced Sca-1+ Tumor Response
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GFP+ MMT tumor cells (106) were inoculated into the right and left mammary fat pads of wild‐type C57BL/6 mice. On day 10, the tumors (about 5 mm3) in the right side received radiation at 9 Gy using XRAD320 (Precision X‐Ray Inc., Branford, CT, USA). The mice were sacrificed on days 3 and 5 after radiation. Then sections of tumor or draining lymph node were prepared, stained with anti‐Sca‐1 antibodies, and examined by light microscopy. Mice were housed and handled in facilities accredited by the American Association for the Accreditation of Laboratory Animal Care. The study was carried out in accordance with the animal protocol approved by the Institutional Animal Care and Use Committee of Boston University Medical Center. The mice were sedated with intraperitoneal administration of ketamine (75 mg·kg−1) and xylazine (5 mg·kg−1), and then, the tumor was irradiated with 9 Gy by XRAD 320 (Precision X‐Ray Inc., N. Branford, CT, USA). The XRAD320 is a self‐contained X‐ray system for delivering a precise radiation dosage to the tumor in small animals. Thus, the systemic exposure of radiation was avoided. The mice were closely followed and would be euthanized if they showed inactivity, ruffled fur coat, or anorexia.
3
Clonal Expansion of Irradiated Tumor Cells
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To determine the clonal expansion of tumor cells upon fractionated irradiation, 100, 300 and 1,000 cells were seeded per well in 6-well plates and irradiated on four consecutive days with a daily dose of 2 gray (Gy) using X-RAD 320 (Precision X-Ray, North Branford, CT USA) or kept untreated as controls. Half of the cells were treated daily with 1 μM 4-Hydroxytamoxifen (TAM, Sigma-Adrich, Germany) or every second day with 30 nM Fulvestrant (Sigma-Adrich, Germany). After 10–14 days in culture, clones were stained with crystal violet and total amount of colonies was quantified as described in [9 (link)]. The survival fraction was computed according to [10 (link)].
4
Generating Mixed Bone Marrow Chimeras
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For generating mixed BMCs, 8- to 10-week-old GF Jh−/− mice were transferred to an autoclaved plastic cylinder sealed with Mylar film. The cylinder-containing GF mice were irradiated with a lethal dose of 900 cGy using X-RAD 320 (Precision X-ray). Irradiated recipient mice were transferred to the isolator and reconstituted with 5 × 106 mixed BM cells. BM cells from Jh−/− mice were mixed with BM cells from either Icosl−/− or H2-Ab1−/− mice in an 8:2 ratio. An 8:2 mixture of Jh−/− BM cells and WT B6 BM cells was transferred into irradiated GF Jh−/− mice as a control. At 6 weeks after reconstitution, BMCs were analyzed. For elucidating the role of long-lived IgE-producing cells in sustained IgE responses, 14-week-old GF B6 mice were similarly irradiated with 900 cGy and reconstituted with BM cells from Rag1−/− mice to deplete lymphocytes.
5
Combination Therapy for Breast Cancer PDX
6
Biodosimetry Laboratory Protocols and Calibration
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Data have been generated by biodosimetry laboratories at Health Canada (HC) and Canadian Nuclear Laboratories (CNL). Blood samples were irradiated by HC with an X-RAD-320 (Precision Xray, North Branford, CT), at CNL with a 137 Cs GammaCell40 (Atomic Energy of Canada Ltd, Ottawa, ON), and processed at both facilities using established protocols (11) . One set of metaphase images from CNL (referred as CNL-low, 1 Gy physical dose) and two sets from HC (referred as HC-low and HC-high, 1 Gy and 3.0-4.0 Gy physical dose, respectively) were used for algorithm development and testing. Images were captured using a Metafer slide scanning platform (Metasystems, Newton, MA). Calibration curves were prepared for samples irradiated at known exposures (0, 0.25, 0.5, 0.75, 1, 2, 3, 4 5 Gy). Samples from previous exercises (designated HS## and CS## in Table 1) were analyzed with these curves before versus after application of matched image selection models. After completing training on ADCI (12) , authors from HC and CNL performed these analyses independently.
7
Multimodal Anti-Cancer Combination Therapy
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Everolimus, BGT226 AND BEZ235 were kindly provided by Novartis (Basel, Switzerland). Vincristine sulfate was purchased from Millennium Pharmaceuticals (Cambridge, MA), Doxorubicin from Pfizer (Melrose Park, NSW, Australia). Ionizing radiation was delivered using an X-ray irradiator (XRAD320, Precision X-Ray, Inc. East Haven, CT) at a dose rate of 0.91 Gy/minute. The following antibodies were purchased from Cell Signaling Technologies (Danvers, MA, USA): rabbit anti-phospho-4E-BP1, rabbit anti-4E-BP1, rabbit anti-phospho-S6RP, mouse anti-S6RP, rabbit anti-phospho-AKT (Ser475), rabbit anti-phospho-AKT (Thr308), rabbit anti-mouse AKT and rabbit anti-LC3B. Rabbit anti-cleaved caspase 3 was purchased from BD Pharmingen, (San Diego, CA, USA).
8
Satellite Cell Engraftment in MDX Mice
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Host MDX mice were anesthetized with isofluorane and hindlimbs were irradiated with 16Gy Xrays delivered at 0.71Gy/min with an X-RAD 320 (Precision X-Ray) biological irradiator. The following day, irradiated MDX mice were implanted subcutaneously with osmotic pumps (Alzet) delivering FK-506 immunosuppressant (LC Laboratories) at 2.5mg/kg/day. Two days after, donor satellite cells from tamoxifen-treated Pax7 CE/+ ;Gli3 +/+ ;R26R YFP and Pax7 CE/+ ;Gli3 fl/fl ;R26R YFP mice were FACS-isolated based on FSC/SSC, lineage negative selection (CD31, CD11b, CD45, SCA1), and positive selection (α7-INTEGRIN, CD34). Donor satellite cells were washed with PBS and resuspended in 0.9% NaCl solution at a concentration of 10 5 cells per 10µl prior to engraftment into the TA muscle of irradiated and immunosuppressed MDX mice. Transplanted TA muscles were collected two weeks after transplantation for measurement of satellite cell engraftment.
9
Photon and Carbon-ion Irradiation Effects
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Photon irradiation was performed by a biological cabinet X-ray irradiator with 320 kV and 12.50 mA (X-RAD 320 Precision X-ray Inc., N. Bradford, Conn.) at single doses of 1 and 2 Gy (110 cGy/min dose rate).
Carbon-ion irradiation was performed at the Heidelberg Ion Therapy Center using the raster scanning technique developed by Haberer et al. [44 ] at the horizontal beam line (Siemens AG). An extended Bragg peak (dose average LET, 103 keV/μm) was adjusted using a 30-mm acrylic shield and discharged single doses of 0.3 and 1 Gy. Cell monolayers were positioned in the middle of the extended Bragg peak.
Irradiation was performed at room temperature 24 hours before the experiments were started, as we previously reported [17 (link)]. For functional assessment of irradiated cells, cell viability was confirmed using trypan blue staining, and only viable cells were used for migration assay.
Assuming a relative biological effectiveness between 2 and 3, we chose single doses of 0.3 and 1 Gy for carbon-ion irradiations to realize the same biological effect as photon irradiation performed with single doses of 1 and 2 Gy.
Carbon-ion irradiation was performed at the Heidelberg Ion Therapy Center using the raster scanning technique developed by Haberer et al. [44 ] at the horizontal beam line (Siemens AG). An extended Bragg peak (dose average LET, 103 keV/μm) was adjusted using a 30-mm acrylic shield and discharged single doses of 0.3 and 1 Gy. Cell monolayers were positioned in the middle of the extended Bragg peak.
Irradiation was performed at room temperature 24 hours before the experiments were started, as we previously reported [17 (link)]. For functional assessment of irradiated cells, cell viability was confirmed using trypan blue staining, and only viable cells were used for migration assay.
Assuming a relative biological effectiveness between 2 and 3, we chose single doses of 0.3 and 1 Gy for carbon-ion irradiations to realize the same biological effect as photon irradiation performed with single doses of 1 and 2 Gy.
10
DNA Damage and Oxidative Stress Modulation
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DNA damage was induced by single exposure (100–500 cGy) using X-ray irradiation (XRT; ionizing radiation; X-RAD 320, Precision X-Ray Inc. North Branford, CT). To increase oxidative stress, cell cultures were supplemented with hydrogen peroxide at varying concentrations (1-100uM) for 30 minutes. To alleviate ROS, cells were treated with 5mM N-acetylcysteine (NAC; Sigma Aldrich) for varying time periods or by culturing cells in low oxygen (1%) (Biospherix hypoxia chamber).
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