Quantifying Tumor-Infiltrating Lymphocytes Densities

Whole slide images were obtained from the CD3, CD4, CD8, FoxP3 and PD-1 immunostaining slides using Panoramic 250 (3DHISTECH, Budapest, Hungary) to quantify the densities of the populations of TILs. The whole tumor area and outer invasive margin of each slide image was labeled using QuPath V.0.1.2.22 (link) The outer invasive margin was defined to be a stromal area 500 µm outside the tumor-stromal interface. The positive cells in the tumor areas were counted using Positive Cell Detection module in the QuPath program. The DAB signal cut-off of each of the immunostaining markers was repeatedly calibrated by the author (HSH). The positive cell densities of the tumor area and outer invasive margin were calculated as the number of positive cells divided by the tumor area measurements (cell counts/mm²) (online supplemental figure 1A–C). We found that the immune cell densities of tumor area and outer invasive margin were strongly correlated (R=0.72–0.93, p<0.001 for all comparisons) (data not shown); therefore, we used the immune cell densities of the tumor area in additional analyses.