Delineation of signaling routes that underlie differences in macrophage phenotypic states

Macrophages represent a major immune cell type in tumor microenvironments, they exist in multiple functional states and are of strong interest for therapeutic reprogramming. While signaling cascades defining proinflammatory macrophages are better characterized, pathways that drive polarization in immunosuppressive macrophages are incompletely mapped. Here, we performed an in-depth characterization of signaling events in primary human macrophages in different functional states using mass spectrometry-based (phospho-)proteomic profiling. Analysis of direct and indirect evidence of kinase activities suggested PAK2 and PKCα kinases as important regulators of <i>in vitro</i> immunosuppressive macrophages. Network integration of these data with the corresponding transcriptome profiles further highlighted FOS and NCOR2 as central transcription regulators in immunosuppressive states. Furthermore, we retrieved single-cell sequencing datasets for tumors from cancer patients and found that unbiased signatures identified here through proteomic analysis were able to separate proinflammatory macrophage populations in a clinical setting and could thus be used to expand state-specific markers. This study contributes to in-depth multi-omics characterizations of macrophage phenotypic landscapes, which could be valuable for assisting future interventions that therapeutically alter immune cell compartments.