We stained 5 µm-thick formalin-fixed paraffin-embedded sections from prostate biopsies and RP specimens using Opal multiplex 6-plex kits, according to the manufacturer’s protocol (PerkinElmer), for a panel of DAPI, CD4, CD8, FOXP3, Ki67, PanCK and PD-L1 (online supplementary methods).
H&E and multiplex immunofluorescence scans were captured by a PerkinElmer Vectra Polaris. Both biopsies and RP tissue slide scans were virtually segmented by a research pathologist into three distinct compartments: CT, tumor IM and NL. Five randomly selected regions of interest (ROI) (mm2, original magnification 40×, 0.25 µm/pixel resolution) from each compartment were then selected from each slide for multispectral imaging (MSI). Vectra Polaris captures spectral information from an MSI image using a multispectral camera, and the intensity of each fluorescent target is extracted from the multispectral data using linear unmixing. Unmixed MSI images were analyzed using inForm OS 2.3.1 software (PerkinElmer). A common algorithm was built based on five representative images, with adjustment from one batch to another due to batch variation. The algorithm consisted of cell segmentation, phenotyping and thresholding. All immune-cell infiltrates were measured as cell counts/mm² of tissue (density).
H&E and multiplex immunofluorescence scans were captured by a PerkinElmer Vectra Polaris. Both biopsies and RP tissue slide scans were virtually segmented by a research pathologist into three distinct compartments: CT, tumor IM and NL. Five randomly selected regions of interest (ROI) (mm2, original magnification 40×, 0.25 µm/pixel resolution) from each compartment were then selected from each slide for multispectral imaging (MSI). Vectra Polaris captures spectral information from an MSI image using a multispectral camera, and the intensity of each fluorescent target is extracted from the multispectral data using linear unmixing. Unmixed MSI images were analyzed using inForm OS 2.3.1 software (PerkinElmer). A common algorithm was built based on five representative images, with adjustment from one batch to another due to batch variation. The algorithm consisted of cell segmentation, phenotyping and thresholding. All immune-cell infiltrates were measured as cell counts/mm² of tissue (density).