Formalin-fixed and paraffin-embedded tumor tissues were cut into 4-µm-thick sections for immunohistochemical (IHC) analysis. After deparaffinization, antigen was retrieved by citric acid buffer (pH 6.0) using a steamer autoclave, then incubated with anti-IDO1 antibody (HPA023072, Sigma, Shanghai, China) at 1:1000 dilution, anti-PD-L1 antibody (ab213524, Abcam, Cambridge, MA, USA) at 1:250 dilution, and anti-CD8 antibody (ab108343, Abcam) at 1:500 dilution overnight at 4 °C. Subsequently, the sections were incubated with a secondary antibody, stained with diaminobenzidine, and then counterstained with hematoxylin, dehydrated, and coverslipped.
All IHC analyses were evaluated by two experienced pathologists (Y.L. and S.L.) who were unaware of patient clinical information. Expression of IDO1 in tumor tissues were considered positive if tumor cytoplasmic and membrane staining >50% regardless of intensity. For PD-L1 staining, >1% tumor membranous staining was considered positive and PD-L1 expression on tumor-infiltrating immune cells was not scored. The quantitative density of CD8+ TILs was evaluated within 5 stromal areas of the tumor under high-power magnification of 400× for each patient. The average number of CD8+ TILs per high power field was counted and the median number of CD8+ TILs was determined as the cut-off point for CD8 density.
All IHC analyses were evaluated by two experienced pathologists (Y.L. and S.L.) who were unaware of patient clinical information. Expression of IDO1 in tumor tissues were considered positive if tumor cytoplasmic and membrane staining >50% regardless of intensity. For PD-L1 staining, >1% tumor membranous staining was considered positive and PD-L1 expression on tumor-infiltrating immune cells was not scored. The quantitative density of CD8+ TILs was evaluated within 5 stromal areas of the tumor under high-power magnification of 400× for each patient. The average number of CD8+ TILs per high power field was counted and the median number of CD8+ TILs was determined as the cut-off point for CD8 density.