Fig. 1: Experimental overview.

a Sample inclusion. From a total of 64 patients, 12 were excluded due technical or quality reasons. Of 52 tumors deemed evaluable, 36 were estimated to have sufficient immune infiltration (> 20 CD45+ cells per ROI (Region of Interest)) for ROI segmentation into tumor (PanCk+ Syto13 + CD45-) and immune (CD45+ Syto13+ PanCk-) AOIs (Areas of Illumination). Following DSP (Digital Spatial Profiling) quantification of antibody-bound probes, 17 AOIs failed QC (quality control) process based low nuclei count, control normalization factor > 3 (indicating low probe binding), or poor segmentation. The final dataset consisted of 156 AOIs from 50 patients, with matched tumor and immune AOIs from 27 patients, and tumor only AOIs from 23 patients. b Specificities of antibodies used for tumor immune microenvironment (TIME) profiling. c Experimental workflow. The DSP technology includes staining FFPE (formalin-fixed paraffin-embedded) samples with panels of fluorescently labelled and barcoded antibodies. Immunofluorescence visualization through scanning is used to guide selection of ROIs. Upon exposure of UV light, barcodes are cleaved off, aspirated and dispensed in a microwell plate. Collected barcodes are hybridized to color-coded probes which are quantified in the nCounter instrument. d ROIs representing insignificant, focal and diffuse immune infiltration were selected from two TMA slides with 3x1mm cores per tumor (red=PanCk, green=CD45, blue=Syto13). Each ROI was segmented into AOIs of tumor (PanCk + , CD45-, Syto13 + , teal masks) and, when possible (> 20 immune cells per ROI), immune (CD45 + , PanCk-, Syto13 + , lime masks), for separate quantitation of biomarkers. Graphs show raw counts of biomarkers for tumor and immune AOIs, for the respective ROIs. e Data was collected from 50 patients, of which 27 had sufficient immune content per ROI for sampling of both immune and tumor AOIs. The number of AOIs collected per patient varied from 1-6.