Inform 2.4.1 image analysis software

Multiplex stained slides were acquired using Vectra® Polaris Quantitative Pathology Imaging System (Perkin Elmer, Boston, MA, USA). Each ×200 multispectral image cube was created by combining images obtained at 10-nm intervals of the emission light spectrum across the range of each emission filter cube. Filter cubes used for multispectral imaging were DAPI (440–680 nm), FITC (520–680 nm), Cy3 (570–690 nm), Texas Red (580–700 nm), and Cy5 (670–720 nm). In each slide, eight to 11 region of interests (ROIs) were selected, and images were spectrally unmixed and segmented (inForm 2.4.1 image analysis software; Perkin Elmer, Wellesley, MA, USA). Data obtained from inForm were sent to Spotfire™ software (TIBCO Software Inc., Palo Alto, CA, USA). Threshold for the positivity of each marker is determined by the pathologist using immunohistochemistry scoring; >0.2 for DOG, >3.0 for CD3, >1.5 for CD8, >1.2 for FoxP3, >1.7 for PD-L1, >0.5 for PD-1, >1.1 for LAG3, >5.0 for Ki-67, >0.7 for CD68, >0.5 for TIM3, and >0.7 for CD204. For each specimen, mean value of the number of cells per mm² in the analysed ROIs was used for further analyses.