Quantifying Immune Cell Markers in Tissue

Immunohistochemistry for CD3 (2GV6) (Roche Diagnostics International AG, Rotkreuz, Switzerland), CD8 (SP57) (Roche Diagnostics International AG, Rotkreuz, Switzerland), and CD68 (KP-1) (Roche Diagnostics International AG, Rotkreuz, Switzerland) was performed on formalin-fixed paraffin-embedded (FFPE) tissues from all 12 cases on a fully automated stainer (VENTANA BechMark ULTRA, Roche Diagnostics International AG, Rotkreuz, Switzerland). The secondary antibodies with horse radish peroxidase (HRP)-3,3′-diaminobenzidine tetrahydrochloride (DAB) were applied as instructed by the manufacturer (See Additional file 3: Table S1 for more details). For each immunohistochemical stain, the number of positive cells per mm² was detected and calculated on QuPath [15 (link)]. Whole slide images were scanned at 20x using a digital slide scanner (APERIO AT2, Leica Biosystems, Nussloch, Germany). Tumor and non-tumor areas of the whole slide image of representative sections for each patient were annotated with QuPath.