Dual Checkpoint Inhibition in M2 Macrophages via Anti-PD-L1 and siRNA-Loaded M1-Exosomes: Enhancing Tumor Immunity through RNA-Targeting Strategies.

The interaction between a cluster of differentiation 47 (CD47) on cancer cells and signal regulatory protein alpha (SIRPα) on macrophages is thought to hinder macrophage phagocytic activity, which can be blocked by combining siRNAs targeting SIRPα (siSIRPα) with simultaneous involvement of activating receptors like FcRs (Fc receptors) anti-programmed death-ligand 1 (anti-PD-L1). For this study, M1 macrophage-derived exosomes were used to deliver the siRNAs, isolated from lipopolysaccharide (LPS)-stimulated RAW264.7 cells and electroporated with siSIRPα. The exosomes were characterized and used to treat M2 macrophages (RAW264.7 cells triggered by interleukin-4 (IL-4)), and the polarization of macrophages was evaluated using flow cytometry, real-time PCR, ELISA, and phagocytosis assays. The anti-tumor functions of treated macrophages were assessed by co-culturing them with 4T1 cells, evaluating the migration and invasion of 4T1 cells, and phagocytosis of 4T1 cells by macrophages. The results showed that siSIRPα-loaded M1-exosomes caused polarization of M2 macrophage toward M1 phenotype and enhanced anti-tumor effects by reducing migration and invasion of 4T1 cells and enhancing phagocytosis of 4T1 cells by macrophages, especially with combination of anti-PD-L1. This study suggests that blocking the SIRPα-CD47 interaction and the PD-1/PD-L1 pathway in M2 macrophages could be a promising therapeutic approach to enhance anti-tumor immune responses.