Vectra 3 pathology imaging system microscope

mIHC/IF was performed (n = 34) using an Opal Multiplex fIHC kit (Akoya Bioscience, Menlo Park, CA, USA), as previously described^{5 (link)}. All cases profiled were also correspondingly profiled on the NanoString PanCancer IO360 panel and included in a combined analysis. Briefly, slides were labeled with PD-L1, CD68, CD8, Foxp3, CD15, and ERG, followed by appropriate secondary antibodies (detailed protocol previously reported^{46 (link)}). Ten images of viable tumor regions that were selected randomly by pathologists were acquired for each case using a Vectra 3 pathology imaging system microscope (Akoya Bioscience, Menlo Park, CA, USA) then analyzed and scored by a pathologist. The number of immune cells scored were normalized against ERG+ (tumor) cells and log2 transformed prior to correlating with NanoString data.

mIHC/IF was performed on FFPE biopsy tissue using Opal Multiplex fIHC kit (Akoya Biosciences, Menlo Park, CA, USA)^{51 (link)}. In brief, 4 um FFPE tissue sections were semi-automatically processed using Leica Bond Max autostainer (Leica Biosystems, Wetzlar, Germany). The slides were stained using 6 primary antibodies (CD8, FOXP3, PD-1, CTLA-4, TCF1 and CD39; summarized in Supplementary Table 5), polymeric HRP-conjugated secondary antibodies from BOND Polymer Refine Detection kit (Cat DS9800; Leica Biosystems, Newcastle, UK), and Opal fluorophore-conjugated tyramide signal amplification (1:100 dilution; Cat #NEL797001KT; Akoya Biosciences, Marlborough, MA, USA). Mounted slides were scanned using Vectra 3 pathology imaging system microscope (Akoya Biosciences). Images were analyzed using inform software (v2.4.6; Akoya Biosciences). Statistical analysis and visualization were performed using GraphPad Prism (v8.0.0; GraphPad Software, Inc., San Diego, CA, USA).