Elective nodal irradiation mitigates local and systemic immunity generated by combination radiation and immunotherapy in head and neck tumors

Lymph nodes from canine patients with head and neck caner were sequenced to determine differences in gene expression after irradiation. RNA was extracted from frozen tumor tissues using Rneasy Plus Mini Kit (QIAGEN) following manufactures protocol. Depending on starting material, samples were eluted in 30-50L RNase-Free water. Samples were initially checked for quantity and purity on a Nanodrop ND-1000 Spectrophotometer (Thermo Fisher) prior to being stored at -80oC until further processing. Samples were additionally quantity and quality checked using the RNA High Sensitivity assays on the Qubit 2.0 Fluorometer (Invitrogen/LifeTechnologies) and 5200 Fragment Analyzer Automated CE System (Agilent), respectively. NanoString gene expression analysis was performed using the Canine IO 360 panel. Nanostring analysis was performed with the nCounter Analysis FLEX system at the University of Arizona Genetics Core. Gene expression count data was analyzed via nSolver software.