A serum-reduced culture system for canine PBMC-derived macrophages enables characterization of M0, M1, and M2 phenotypes and tumor-associated interactions.

Canine macrophages are increasingly recognized as important regulators of tumor immunity; however, in vitro culture systems that minimize serum-derived variability remain limited. In this study, we established a serum-reduced culture system for canine peripheral blood mononuclear cell (PBMC)-derived macrophages and characterized their phenotypic, transcriptional, and functional properties. PBMCs from healthy Beagle dogs (n = 3) were differentiated into M0, M1, and M2 macrophages using Macrophage Base Medium supplemented with canine serum or recombinant cytokines. Morphology was assessed by phase-contrast microscopy, macrophage identity was evaluated by Iba1 immunostaining and AIF1 expression, and exploratory transcriptomic profiles were examined by RNA sequencing (n = 1). Phagocytic activity was assessed using FITC-labeled latex beads and flow cytometry, and macrophage-tumor cell interactions were evaluated by co-culture with canine urothelial carcinoma cells. M0, M1, and M2 macrophages exhibited distinct morphologies and transcriptional patterns. M1 macrophages showed increased expression of inflammatory genes, including IL1B and IFNG, whereas M2 macrophages showed increased expression of genes associated with alternative activation and immune regulation, including CD209, CD5L, and IL33. Phagocytic activity was observed in all subsets. In co-culture experiments, macrophages exhibited polarization-dependent interaction patterns with tumor cells, with M2 macrophages exhibiting features reminiscent of tumor-associated macrophages. These findings provide a pilot evaluation of this serum-reduced culture system, which generates phenotypically distinct canine macrophage subsets and may serve as a useful in vitro platform for future studies of canine macrophage polarization and tumor-associated immune responses.