Fig. 1

Creation and analysis of IIC-enriched biopsy-based NSCLC TMA. a Illustration depicting TMA creation workflow. Baseline biopsies from a NSCLC patient cohort (n = 81) were paraffin embedded, and cut sections were stained using α-CD45 to demarcate IIC-dense regions then selected for TMA construction using original blocks. Cut sections from resulting TMA were then stained using MP-IF panels targeting immune-related antigens including ICPs and IIC subsets. Slides were scanned to create super images permitting the development of algorithms computing antigens of interest and their colocalization for normalization (figure elements modified from Servier medical art). b Image representing α-CD45 IHC stained biopsies defining IIC-dense areas. c Example of MP-IF panels demonstrating α-ICP (green), α-CD3 (pink), α-CD4 (red), and α-CD8 (yellow) antibodies validated to surround DAPI-staining nuclei (blue). IIC-enriched core selection was performed by two different operators. TMA cores were randomized and TMAs were created by two operators. HRP, horseradish peroxidase; 2° ab, secondary antibody; AF, Alexa-Fluor dye; α, anti; μm, micron; mm, millimeter