Multiplex Immunofluorescence Analysis of PD-L1, Immune Cells, and Tumor Microenvironment

PD-L1 expression was detected using the PD-L1 IHC 22C3 pharmDx assay (Agilent Technologies, Santa Clara, CA, USA) and was assessed by combined positive score (CPS), where CPS ≥1 was considered as positive. The mIF staining was performed using PANO 7-plex IHC kit (Panovue, Beijing, China), according to the manufacturer’s instructions as described previously (17 (link)). Briefly, CD8 marker was used to identify T cells. The natural killer (NK) cells were divided into CD56dim (weak staining) and CD56bright (strong staining) according to the intensity of membrane staining by CD56 antibody. Tumor-associated macrophages (TAMs) were identified by CD68 and HLA-DR and were divided into TAM1 (CD68⁺ and HLA-DR⁺) and TAM2 (CD68⁺ and HLA-DR⁻). S100 staining was used to define the tumor center and the invasive margin. The stained slides were scanned and built a single stack image subsequently by the Mantra System (PerkinElmer, Waltham, MA, USA). The reconstruction of images was performed using inForm image analysis software (PerkinElmer) for multispectral unmixing to remove autofluorescence.