Quantitative Analysis of Dendritic Cells in Tumor Microenvironment

Quantitative analysis of immunohistochemical reactions was performed using light microscopy, and the numbers of positively stained cells for each antibody were obtained. Slides were initially examined at a low magnification (10× lens) to select the areas that were most abundant in positively identified DCs (so-called hot spots). Within the chosen areas, the counts of positively stained DCs in five representative fields of view were summed up at a high magnification (40× lens) and expressed per 1 mm². An assessment of cells’ morphology preceded establishing their numerosity: a cell was considered a positively stained DC if it had a visible nucleus, dendritic appearance (e.g., cytoplasmic processes), and intensely colored cytoplasm at 40× magnification. Analyses of each monoclonal antibody’s reactivity were performed separately for the tumor stroma, glandular endothelium, and invasive border (Figure S2 in the Supplementary Materials). The latter term refers to the field of view comprising the cancerous margin and the adjacent healthy tissues in an even ratio. Samples were also evaluated for the presence of co-occurrent non-neoplastic endometrium. Numbers of positively identified DCs per mm² in stroma, glands, and margins of healthy tissue were utilized to create an internal positive control group (Figure S3 in the Supplementary Materials). Two authors performed the pathological analyses separately and solved any disagreements via discussion.