Digital image analysis software

Tumor tissues from the two-tumor model of LLC were fixed in 10% neutral buffered formalin, processed, embedded in paraffin, and sectioned (4 μm). For multiplex staining, a board-certified veterinary pathologist reviewed the hematoxylin and eosin-stained slides and marked areas of immune cell infiltration, after which 6-mm tissue cores, including peripheral and central areas of the tumor section, were manually incorporated into a tissue microarray. A Leica Bond Rx Auto Stainer with antibodies (STAT6 and IBA-1) listed in Supplementary Table S2 and a Vector TrueVIEW Autofluorescence Quenching Kit were used for multiplex staining per the manufacturer’s instructions. Microarrays were imaged at 20X with a Leica Versa 8 fluorescent digital scanning microscope system, with Leica digital image analysis software used for quantification. For image pre-processing, tissue cores were each annotated separately, and the exclusion tool was used to exclude histologic artifacts. Four 500×500 μm areas were placed at the periphery and central areas of the tumor. A cellular immunofluorescence algorithm from Leica Imagescope software version 12.4.6 was trained for quantification of markers in each channel.