Multiplex Immunofluorescence Staining of Tissue Sections

The immunofluorescence was performed at the Molecular Cytology Core Facility of Memorial Sloan Kettering Cancer Center using Discovery XT processor (Ventana Medical Systems). The tissue sections were deparaffinized with EZPrep buffer (Ventana Medical Systems), antigen retrieval was performed with CC1 buffer (Ventana Medical Systems). Sections were blocked for 30 min with Background Buster solution (Innovex), followed by avidin-biotin blocking for 8 min (Ventana Medical Systems). Multiplex immunofluorescence stainings were performed as previously described^{53 (link)} (see Supplementary Note). Stained slides were digitized using Pannoramic Flash 250 (3DHistech, Hungary) using 20x/0.8NA objective. Regions of interest were drawn on the scanned images using Pannoramic Viewer (3DHistech, Hungary) and exported into tiff images. ImageJ/FIJI was used to segment DAPI-stained nuclei and count the cells with positive signal. Ki67 was quantified according to the recommendations for breast cancer^{54 (link)}, where the percentage of positively stained nuclei is quantified among the total number of malignant cells as previously described¹³ .