To study the protein expression profile and the relationship of CMTM6/4 and PD-L1 in patients with GC, we applied multiple immunofluorescence staining (MIS) by using multiplexed Opal fluorophores that can simultaneously evaluate six markers including CMTM6, CMTM4, PD-L1, PD-1, cytokeratin (CK), and DAPI (nuclear stain). This evidence concerns the gene CD274 and gastric cancer.