Elimination of Cancer Cells in Co-Culture: Role of Different Nanocarriers in Regulation of CD47 and Calreticulin-Induced Phagocytosis

Under healthy conditions, pro- and anti-phagocytic signals are balanced. Cluster of Differentiation 47 (CD47) is believed to act as an anti-phagocytic marker that is highly expressed on multiple types of human cancer cells including acute myeloid leukemia (AML) and lung and liver carcinomas, allowing them to escape phagocytosis by macrophages. Downregulating CD47 on cancer cells discloses calreticulin (CRT) to macrophages and recovers their phagocytic activity. Herein, we postulate that using a modified graphene oxide (GO) carrier to deliver small interfering RNA (siRNA) CD47 (CD47_siRNA) in AML, A549 lung, and HepG2 liver cancer cells in co-culture in vitro will silence CD47 and flag cancer cells for CRT-mediated phagocytosis. Results showed a high knockdown efficiency of CD47 and a significant increase in CRT levels simultaneously by using GO formulation as carriers in all used cancer cell lines. The presence of CRT on cancer cells was significantly higher than levels before knockdown of CD47 and was required to achieve phagocytosis in co-culture with human macrophages. Lipid nanoparticles (LNPs) and modified boron nitride nanotubes (BNPs) were used to carry CD47_siRNA, and the knockdown efficiency values of CD47 were compared in three cancer cells in co-culture, with an achieved knockdown efficiency of >95% using LNPs as carriers. Interestingly, the high efficiency of CD47 knockdown was obtained by using the LNPs and BNP carriers; however, an increase in CRT levels on cancer cells was not required for phagocytosis to happen in co-culture with human macrophages, indicating other pathways' involvement in the phagocytosis process. These findings highlight the roles of 2D (graphene oxide), 1D (boron nitride nanotube), and "0D" (lipid nanoparticle) carriers for the delivery of siRNA to eliminate cancer cells in co-culture, likely through different phagocytosis pathways in multiple types of human cancer cells. Moreover, these results provide an explanation of immune therapies that target CD47 and the potential use of these carriers in screening drugs for such therapies in vitro.