Software‐assisted morphometric and phenotype analyses of human peripheral blood monocyte‐derived macrophages induced by a microenvironment model of human esophageal squamous cell carcinoma

Human macrophages play important roles in tumor promotion and are called tumor‐associated macrophages (TAMs). We previously demonstrated that human esophageal squamous cell carcinomas (ESCCs) contain TAMs and that these TAMs tend to have tumor‐supporting features. Here we exposed human macrophages to conditioned media of TE‐series human ESCC cell lines (TECMs) to generate an ESCC extracellular stimuli‐influenced TAM model. CD14⁺ peripheral blood monocytes (PBMos) from healthy donors were treated with M‐CSF and with additional IL‐4 or TECM exposure. Morphological changes of the cells and the induction of CD163/CD204 proteins were detected in the TECM‐exposed model TAMs by immunofluorescence. A software‐assisted immunofluorescent cell image analysis showed increased CD163/CD204 positivity in the model TAMs and a weak to moderate positive correlation between the cytoplasmic area and the sum fluorescent intensity of CD204. Morphological changes of the cells were significantly reflected by several cytomorphometric parameters. PBMos were elongated with M‐CSF treatment, then enlarged with TECM exposure. The cytoplasmic aspect ratio was decreased by M‐CSF treatment and slightly increased by TECM exposure. The nuclear‐cytoplasmic ratio decreased during the whole process of cell differentiation. This system is useful for quantitative assessments of TAM‐like morphological changes of macrophages and the induction of CD163/CD204 in a model ESCC microenvironment.