Blockade of CD47-Signaling Regulatory Protein alpha Signaling Enhances the Macrophage Phagocytic Activity Against Cancer Cells.

Abstract

Background: Liver transplantation (LT) is the best treatment for unresectable, early-stage hepatocellular carcinoma (HCC). However, HCC recurrence is common, due to either occult metastasis development or circulating tumor cell engraftment. We previously demonstrated that the phagocytic activity of human liver macrophages is inhibited by the binding of signaling regulatory protein alpha (SIRPα) on macrophages to CD47 on target cells, a major regulator of phagocytic responses (2006 PNAS). Using in vitro and in vivo mouse syngeneic models, we examined whether the inhibition of CD47-SIRPα signaling would increase macrophage phagocytic activity against cancer cells. Methods: For the in vitro phagocytosis assay, Hepa1-6 and CMT93 target cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and incubated with mouse peritoneal cavity (PerC) macrophages and either anti-mouse SIRPα-blocking monoclonal antibodies (mAbs) (SIRPα mAbs) or isotype-matched control antibodies (isotype Abs). The phagocytosis of CFSE-labeled target cells was measured by flow cytometry. For the in vivo phagocytosis assay, CFSE-labeled target cells were injected into the peritoneal cavity, together with SIRPα mAbs, CD47 mAbs, or isotype Abs. CD47 knock-down (KD) was achieved via lentiviral particle-mediated shRNA delivery to Hepa1-6 cells, which were used in both in vitro and in vivo assays. Results: In the in vitro phagocytosis assay, anti-SIRPα mAb addition markedly enhanced the phagocytic activity of PerC macrophages against Hepa1-6 and CMT93, compared with isotype Abs treatment (p < 0.01). In the in vivo phagocytosis assay, a significant increase in phagocytic activity (p < 0.01) was observed with SIRPα mAb treatment, but not with CD47 mAb treatment. CD47 KD Hepa1-6 cells were significantly more sensitive to macrophage phagocytosis than parental Hepa1-6 cells in both in vitro and in vivo assays (p = 0.01), demonstrating the pivotal role of the CD47-SIRPα interaction in restricting tumor cell killing. In vivo CD47 mAb administration led to a remarkable decrease in CD80, CD86, and MHC class II expression on macrophages, implying their functional impairment. This finding might explain why treatment with CD47 mAbs alone did not enhance macrophage phagocytic activity against cancer cells. Conclusions: CD47-SIRPα interaction could be a therapeutic target for preventing HCC recurrence in LT recipients.