Dual blockade of GSTK1 and CD47 improves macrophage-mediated phagocytosis on cancer cells.

CD47 is a crucial anti-phagocytic signal in regulating macrophage responses and its manipulation offers the therapeutic potential in cancer treatment. However, in many cases, blockade of CD47 by itself is insufficient to activate macrophage effectively, indicating other unidentified phagocytosis-regulating factors to resist the macrophage activity. In this study, a genome-wide human CRISPR-Cas9 library was developed for comprehensive screening of phagocytosis-regulating factors in the context of CD47 blockade. The screening results identified GSTK1 as a potential anti-phagocytic signal counteracting the efficacy of CD47-based phagocytosis. The disruption of GSTK1 significantly increased the phagocytosis rate of cancer cells by macrophages in combination with anti-CD47 antibody. Further mechanism investigation unveiled that GSTK1 blockade increased the membrane exposure of calreticulin in different cancer cells, which might be the primary mechanism driving enhanced macrophage-mediated phagocytosis. To this end, siGSTK1-loaded nanoparticles (siGSTK1-LNPs) were designed to suppress the GSTK1 expression efficiently. The comparable phagocytosis efficacy was also observed when combining siGSTK1-LNPs with anti-CD47 antibody. Above all, GSTK1 blockade was identified as a promising and feasible stimulus for enhancing the effectiveness of anti-CD47 antibody, introducing a novel and effective combination approach in cancer immunotherapy.