NanoString
Expression of genes belonging to several immune-related signatures was assessed by a custom-designed NanoString nCounter multiplex CodeSet enabling the determination of 364 genes. The gene signatures were selected for providing information on B-cell content and differentiation, TLS formation, follicular T helper cells, TEX subsets, tumor-associated endothelial cells, ICB response, and guadecitabine-specific gene upregulation. For NanoString experiments, panel probes (capture and report) and 200 ng of RNA were hybridized overnight at 65 °C for 16 h. Samples were scanned at maximum scan resolution capabilities (555 FOV) using the nCounter Digital Analyzer. Quality control of samples, data normalization, and data analysis were performed using nSolver software 4.0 (NanoString Technologies).