Poly(I:C) stimulation is superior than Imiquimod to induce the antitumoral functional profile of tumor-conditioned macrophages

Macrophage plasticity is the ability of mononuclear phagocytes to change phenotype, function, and genetic reprogramming upon encounter of specific local stimuli. In the tumor microenvironment, Tumor-Associated Macrophages (TAMs) acquire an immune- suppressive and tumor-promoting phenotype. With the aim to re-educate TAMs to anti- tumor effectors, in this study, we used two immunestimulatory compounds: the TLR7 agonist Imiquimod (IMQ) and the TLR3 agonist Poly(I:C). To better mimic in vitro the response of TAMs, we used Tumor-Conditioned Macrophages (TC-Mφ) differentiated in the presence of tumor cell supernatants. Our results show that TC-Mφrespond differently from conventional M2-polarized macrophages. Upon stimulation with IMQ, TC-Mφdid not upregulate major histocompatibility complex (MHC II) molecules and unexpectedly expressed increased CD206. With both compounds, TC-Mφproduced higher levels of inflammatory cytokines than M2 macrophages. IMQ and Poly(I:C) differed in the types of regulated genes and secreted mediators. Reflecting their signaling pathways, only IMQ significantly induced IL-1βand IL-6, while only Poly(I:C) stimulated CXCL10, and both upregulated CCL5. Of note, using a novel cytotoxicity assay, Poly(I:C), but not IMQ, was effective in triggering the cytotoxic activity of TC-Mφagainst cancer cells. Overall, the results demonstrate that Poly(I:C) stimulation of TC-Mφis superior than IMQ in terms of macrophage re-education toward antitumor effectors.