Selective nodal radiation may be a more effective approach in cancer treatment CUAnschutz NatureComms
Radiation was delivered using an X-RAD SmART irradiator with a beam energy of 225 kVp to mice under isoflurane anesthesia. Treatment planning and Monte Carlo simulations were performed using SmART-ATP software . Radiation doses were 24 Gy in three fractions for tumor and 15 Gy in three fractions for ENI.
For HNSCC models, buccal tumors were treated using a tangent beam positioned to not intersect a pair of opposed lateral beams used for nodal irradiation. For the 4T1 breast cancer model, mice were placed in a lateral recumbent position with the tumor gently pulled away from the chest using a plastic ring. A 1 cm circular beam was then aligned using fluoroscopy to irradiate the tumor while hitting minimal normal tissue.
For sentinel lymph node irradiation, mice were irradiated with a fractionated dose of 8 Gy × 3 using the XRAD SmART Irradiator . Irradiation of the primary tumor and sentinel lymph nodes was done by angling the beam to 325 degrees and positioning it such that the tumor and lymph node were irradiated while avoiding as much normal tissue as possible, as well as other lymph nodes. The presence of the sentinel node in the irradiated field was confirmed in fluoroscopy using 2.
After surface staining, cells were fixed and permeabilized using the Foxp3 perm/fix kit overnight. Following incubation, cells were stained for intracellular markers and incubated for 30 min. Samples were then run on a Cytek Aurora spectral cytometer at the University of Colorado Diabetes Research Center Flow Cytometry Core. Fluorescence minus one controls were used to determine gating strategy. Flowjo analysis software and Cytobank was used for data analysis.
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