RNAi screen in apoptotic cancer cell-stimulated human macrophages reveals co-regulation of IL-6/IL-10 expression

Tumor-associated macrophages (TAM) are a major supportive component within neoplasms and are characterized by a plethora of functions that facilitate tumor outgrowth. Mechanisms of macrophage attraction and differentiation to a tumor-promoting phenotype, defined among others by distinct cytokine patterns such as pronounced interleukin (IL-10) production, are ill-defined. We aimed to identify signaling pathways that contribute to the generation of TAM-like macrophages using an adenoviral RNAi-based approach. Primary human monocyte-derived macrophages were stimulated with apoptotic tumor cell supernatants (ACM) to induce a TAM-like phenotype, characterized by secretion of IL-10, IL-6, IL-8 but repression of IL-12. For the high-throughput screen, macrophages were transduced with 8495 constructs of the adenoviral shRNA SilenceSelect® library of Galapagos BV, which aims at identifying druggable targets. We identified 96 genes involved in IL-10 production in response to ACM and observed a pronounced cluster of targets regulating both IL-10 and IL-6. Validation of five targets within the IL-10/IL-6 cluster was performed using siRNA or pharmacological inhibitors in human primary macrophages. Among those, interleukin 4 receptor-α and cannabinoid receptor 2 were confirmed as regulators of IL-10 and IL-6 secretion by ACM-stimulated macrophages. Our approach characterizes cellular functions of transfection-resistant, highly plastic and versatile cells and identifies novel targets involved in the generation of a TAM-like phenotype in human macrophages.