Multiplex immunofluorescence
Selected samples were assessed by multiplex immunofluorescence with antibodies against cytokeratin (Zsbio, clone number: AE1/AE3, 1:200 dilution), CD8 (Abcam, clone: C8/468 + C8/144B, dilution: 1:200), FoxP3 (Abcam, clone: 236A/E7, dilution: 1:100), CD68 (Abcam, clone: KP1, dilution: 1:100), PD-1 (Zsbio, clone: UMAB199, dilution: prediluted), and PD-L1 (Cell Signaling Technology, clone: E1L3N, dilution: 1:200). The staining was performed using the Opal 7-Color IHC Kit (Akoya Biosciences, USA) and imaged by a PerkinElmer Vectra 3.0 (Perkin Elmer, Hopkington, MA) multispectral microscope. Specificity for each staining has been validated.