Tumor-associated macrophages are widely recognized for their importance in guiding pro-tumoral or antitumoral responses. Mediating inflammation or immunosuppression, these cells support many key events in cancer progression: cell growth, chemotaxis, invasiveness, angiogenesis and cell death. The communication between cells in the tumor microenvironment strongly relies on the secretion and recognition of several molecules, including damage-associated molecular patterns (DAMPs), such as adenosine triphosphate (ATP). Extracellular ATP (eATP) and its degradation products act as signaling molecules and have extensively described roles in immune response and inflammation, as well as in cancer biology. These multiple functions highlight the purinergic system as a promising target to investigate the interplay between macrophages and cancer cells. Here, we reviewed purinergic signaling pathways connecting cancer cells and macrophages, a yet poorly investigated field. Finally, we present a new tool for the characterization of macrophage phenotype within the tumor. Image cytometry emerges as a cutting-edge tool, capable of providing a broad set of information on cell morphology, expression of specific markers, and its cellular or subcellular localization, preserving cell-cell interactions within the tumor section and providing high statistical strength in small-sized experiments. Thus, image cytometry allows deeper investigation of tumor heterogeneity and interactions between these cells. ? 2020 International Society for Advancement of Cytometry.