Automated Multiplex Immunofluorescence Staining

The validation pipeline and details of the development of the multiplex immunolabeling protocol have been described previously by our group and are shown in supplementary material, Figure S1 and Supplementary materials and methods [15 (link), 16 (link)]. Multiplex immunofluorescence staining was performed using the LabSat® Research platform (Lunaphore Technologies, Tolochenaz, Switzerland), a fully automated tissue‐staining instrument for rapid immunostaining which utilizes a microfluidic technology for the rapid and uniform delivery of reagents to tissue samples [17 (link)]. In brief, each TMA section was subjected to six successive, automated rounds of antibody staining, including pan‐cytokeratin, CD8, CD68, FOXP3, PD‐1, and PD‐L1. Nuclei were counterstained with spectral DAPI (Akoya Biosciences, Marlborough, MA, USA). A full protocol is provided in Supplementary materials and methods. TMAs were scanned using a PhenoImager HT Automated Quantitative Pathology Imaging System (Akoya Biosciences). Image analysis was performed using the inForm software framework (version 2.4.8, Akoya Biosciences), shown in Supplementary materials and methods.