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 a strong interest for therapeutic reprogramming. While signaling cascades defining pro-inflammatory macrophages are better characterized, pathways that drive polarization in immunosuppressive macrophages are incompletely mapped. We performed an in-depth characterization of signaling events in primary human macrophages in different functional states using mass spectrometry-based proteomic and phosphoproteomic profiling. Analysis of direct and indirect footprints of kinase activities has suggested PAK2 and PKCα kinases as important regulators of better studied in vitro immunosuppressive macrophages (IL-4/IL-13 or IL-10 stimulated) and has highlighted several other major regulatory kinases in less well studied macrophages with immunosuppresive roles. Network integration of these data with the corresesponding transcriptome profiles has, among others, 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 the unbiased signatures identified through proteomic analysis were able to successfully separate different 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.