Inform image analysis software

To evaluate the densities of IL-25⁺, CD4⁺, CD8⁺, CD103⁺ and Foxp3⁺ cells, areas of intra-tumor (IT) and non-tumor (NT) tissue sections were screened by microscopy at low magnification (× 100); five representative spot images were then selected and captured at × 200 magnification using a computerized system that included a Digital Sight DS-Fi1 camera installed on a Nikon Eclipse 80i light microscope (Nikon). Cells that were stained positive were automatically counted using inForm image analysis software (Perkin-Elmer Applied Biosystems; Hopkinton, MA, USA) [48 (link)]. Cell numbers were expressed as mean ± SEM per mm².

To assess the expression level of GLUT1/ASCT2, the Vectra-Inform image analysis system (Perkin-Elmer Applied Biosystems) was used as described in previous studies [18 (link), 19 (link)]. In brief, a scanning protocol was designed on the basis of the TMA core number and size. Nuance multispectral image cubes (8-bit) were acquired from the TMA at a constant 200× magnification. The spectral library containing two chromogens was built with two control HCC tissue slides stained with only 1 chromogen (3,3′-diaminobenzidine or hematoxylin). The spectral library was used to unmix the signals on the multicolored images by recognizing their unique spectral curves for quantitation. Images were analyzed using InForm image analysis software (version 2.0.1; Perkin-Elmer Applied Biosystems) to identify tissue compartments (tumor and non-tumor tissue) and subcellular compartments (nucleus, cytoplasm, and membrane). The H-score was calculated by quantifying target signals in selected tissues and cellular compartments of interest following the manufacturer’s protocol.