NanoString gene expression analysis

C57BL/6 mice were implanted with stereotactic injection of 50,000 GL261 cells as described above. On day 14 after tumor implantation, 10 mice in each group were treated with either PBS or LIPU. On day 16 after tumor implantation, mice were euthanized and brains were collected after cardiac perfusion with PBS. Percoll density gradient centrifugation was then used for immune cell isolation from the hemisphere of tumor implantation. Immune cells underwent mRNA extraction (RNeasy Plus Mini Kit, Quiagen, Germantown, MD) for NanoString analysis. Analysis of mRNA was performed with the nCounter PanCancer Immune Profiling Panel (NanoString Technologies, Inc., Seattle, WA). Sample preparation and hybridization was performed as described previously (16). Target molecules were quantified by imaging immobilized fluorescent reporters using the nCounter Digital Analyzer. mRNA gene expression of 770 immune-related genes were analyzed using the nSolver 4.0, R version 3.3.2, and Advanced Analysis 2.0 software. The genes specific to functional signaling pathways and immune cell types were categorized based on the attached manual.