Multiplex immunofluorescence staining
Multiplex immunofluorescence staining was performed using Ultivue FixVUE 8-plex multiplex immunostaining kit (CD3, CD4, CD8, FoxP3, granzyme B, Ki67, PanCK/Sox10 and PD-L1) and conducted on a Leica Bond Rx (Leica Biosystems) fully automated immunostainer, according to the kit protocol. The whole slides were scanned using an AxioScan.Z1 microscope slide scanner; images were stacked using Ultivue’s Ultistacker software for InSituPlex image co-registration (version 1.0); and stacked image datasets were analyzed using HALO image analysis software. Images were annotated and analyzed using High Plex FL version 3.2.1 algorithm (Indica Labs), resulting in co-localization output for key immunophenotypes, such as CD4 T cells (CD3