Vectra slide scanner

For multiplex staining, we followed the Opal protocol staining method36 for the following markers: CD73 (1:200, Abcam, ab91086) with subsequent visualization using fluorescein Cy3 (1:50); CD163 (1:25, Leica Biosystems, NCL-L-CD163) with visualization accomplished using Cy5 (1:50); and CD68 (1:100, Dako, M0876) with visualization using Cy5.5 (1:50). Nuclei were subsequently visualized with DAPI (1:2000). All of the sections were cover-slipped using Vectashield H-1400 mounting media. For multispectral analysis, a detailed methodology was followed as described previously (Stack et al., 2014). Each of the individually stained sections was utilized to establish the spectral library of fluorophores required for multispectral analysis. The slides were scanned using the Vectra slide scanner (PerkinElmer) under fluorescent conditions. For each marker, the mean fluorescent intensity per case was then determined as a base point from which positive cells could be established. Finally, the co-localization algorithm was used to determine percent of CD68, CD163 and CD73 staining.

Analysis using the Vectra system was performed as described in previous studies [5 (link),13 (link)]. Stained slides were loaded into the Vectra slide scanner (PerkinElmer, Waltham, MA, USA), and a scanning protocol was produced. Eight-bit Nuance multispectral image cubes were created with the ×20 lens. Nuance software (version 3.0.0; PerkinElmer) was then used to build the spectral library (Fig. 1). Three control slides with 1 chromogen (3,3′-diaminobenzidine, hematoxylin, and Warp Red) were scanned, and spectral curves were defined to unmix signals (Fig. 2).
Images were imported using inForm software (version 1.4; PerkinElmer). Initially, 18% of cores from the TMA were used to set up an algorithm for differentiation, assuring 97% acceptable tissue segmentation [5 (link)]. Target signals were then quantified in selected tissues and cellular compartments of interest (Fig. 3). For HIF1α and HIF2α, nuclear expression was used in the analysis because these transcription factors are active when present in the nuclear compartment. For Ki-67, the percentage of cells with positive nuclear staining was used because Ki-67 is a cellular marker for proliferation located in nuclei. For CRP, the global cellular expression was used in the analysis. Core images with less than 5% epithelial component, significant folding, or loss of tissue were excluded.

Multiplex staining was performed per the Opal protocol staining method ^{15 (link)} for markers: CD4 (1:25, CM153BK, Biocare) with subsequent visualization using fluorescein AF-488 (1:50); CD8 (1:200, M7103, Dako) with visualization using AF-594 (1:50); CD68 (1:100, M0876, Dako) with visualization using AF-647; VISTA (1:100, Janssen) with visualization using coumarin (1:50); CD163 (1:100, NCL-L-CD163, Leica) with visualization using AF-488 and PD-L1 (1:100, 13684, Cell Signaling Technology) with visualization using AF-555 (1:50). Nuclei were visualized with DAPI (1:2000). All sections were cover-slipped using Vectashield Hardset 895 mounting media.
For multispectral analysis, each individually stained section (CD4/AF-488, CD8/AF-594, CD68/AX-647, VISTA/coumarin, PD-L1/AF-555, CD163/AF-488, and DAPI) was utilized to establish the spectral library of fluorophores. Slides were scanned using the Vectra slide scanner (PerkinElmer). For each marker, the mean fluorescent intensity per case was determined as a base point from which positive calls could be established. The co-localization algorithm was used to determine percent of PD-L1 and VISTA staining on each cellular subset. Five random areas on each sample were analyzed blindly by a pathologist at 20× magnification.