title: RNAi screen in apoptotic cancer cell-stimulated human macrophages reveals co-regulation of IL-6/IL-10 expression.
abstract: 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.
authors.affiliation: Goethe-University Frankfurt, Institute of Biochemistry I/ZAFES, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Ley
authors.forename: Andreas
authors.initials: A
authors.lastname: Weigert
authors.forename: Jean-Karim
authors.initials: JK
authors.lastname: Hériché
authors.forename: Blandine
authors.initials: B
authors.lastname: Mille-Baker
authors.forename: Richard A J
authors.initials: RA
authors.lastname: Janssen
authors.forename: Bernhard
authors.initials: B
authors.lastname: Brüne
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000951:Antigens, Neoplasm / Q000276:immunology; D002478:Cells, Cultured; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics / Q000276:immunology; D057166:High-Throughput Screening Assays; D006801:Humans; D016753:Interleukin-10 / Q000235:genetics / Q000378:metabolism*; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism*; D008264:Macrophages / Q000276:immunology*; D058990:Molecular Targeted Therapy; D009369:Neoplasms / Q000276:immunology*; D061251:Primary Cell Culture; D034741:RNA, Small Interfering / Q000235:genetics*; D043885:Receptor, Cannabinoid, CB2 / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism; D019139:Tumor Escape / Q000235:genetics / Q000276:immunology; D059016:Tumor Microenvironment / Q000235:genetics / Q000276:immunology; D053662:Interleukin-4 Receptor alpha Subunit
chemical_list: D000951:Antigens, Neoplasm; D016753:Interleukin-10; D015850:Interleukin-6; D034741:RNA, Small Interfering; D043885:Receptor, Cannabinoid, CB2; D011956:Receptors, Cell Surface; C571431:Cnr2 protein, mouse; C504002:Il4ra protein, mouse; D053662:Interleukin-4 Receptor alpha Subunit