Inform 2.4.8 image analysis software

To assess the consistency of the staining markers with the mIF panel over time the TMA was staining with the on consecutive sections at three time points with a 1-week interval (week 1, week 2 and week 3), the 36 cores contained in the TMA from the 12 MPM cases were stained in the autostainer Leica Bond RX and scanned at the three time points using the Vectra/Polaris 3.0.3 multispectral imaging system (Akoya Biosciences) through the full emission spectrum from 440 to 780 nm, to extract fluorescence intensity information from the images using positive tonsil controls from each run staining to calibrate the spectral image scanner protocol at 20 × magnification (0.5 µm/pixel). To determine consistence of the staining across the time each marker was quantify individually using a spectral signature for each fluorophore obtained by the “spectral unmixing library” using the the same algorithm from the InForm 2.4.8 image analysis software (Akoya Biosciences). The percentages representing for each marker were calculated by dividing the absolute number of each marker by the absolute number of total nucleated cells (DAPI +) on each core at each time point.

A spectral library was created for multispectral image analysis visualization and fluorophore extraction. Control tissues were stained using a CD20 antibody (B-cell marker, clone L26, dilution 1:100, Dako) as an abundant expression marker in the tonsil linked to one of the eight Opal fluorophore tyramides, following conditions similar to those used for IHC but without DAPI to obtain abundant signal with each of the fluorophores (Supplementary Fig. 1)^{19 (link)}.Figure 1

Microphotographs of representative examples of multiplex immunofluorescence (mIF) and cord plots of cells co-expression of makers in the different immuno-oncology panels in the NSCLC cohort. For each panel, the composite mIF images (left) and the diversity of cell phenotypes by the markers co-expressed (right) are shown. mIF 20 × magnification. The images were generated using Vectra-Polaris 3.0.3 scanner system and InForm 2.4.8 image analysis software (Akoya Biosciences) and R studio software version 3.6.1.