P06.07 Targeting immune checkpoint proteins CD47 and galectin-3 and -9 to improve macrophage mediated phagocytosis of pancreatic tumor cells

Background Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal tumor entities, with steadily increasing incidence. The tumor microenvironment (TME) is characterized by an intensely immunosuppressive stroma, which comprises up to 90% of the tumor volume. Tumor-associated macrophages (TAMs) are the dominant immune cell population in PDAC and mostly exhibit a M2-like phenotype supporting the immunosuppressive milieu as well as tumor progression. Similar to several entities, the immune checkpoint molecule CD47 is upregulated in PDAC and correlates with advanced disease stages and poor outcomes. CD47 mediates a ‘don’t eat me’ signal to macrophages after binding to signal regulatory protein α (SIRPα), serving as an immune escape mechanism by evading phagocytosis. Additionally, lectins like galectin-3 and -9 are emerging as potential immune checkpoint targets as they are promoting tumor progression and immunosuppression by impacting immune cell differentiation and activity. Therefore, this study aimed at targeting CD47 as well as galectin-3 and -9 to improve PDAC cell phagocytosis by macrophages.Methods Monocytes were isolated from peripheral blood of healthy donors and differentiated into M1- and M2-like macrophages. Multiple PDAC cell lines as well as M1- and M2-like macrophages were characterized regarding their immune checkpoint expression and localisation. PDAC cell phagocytosis by M1- or M2-like macrophages was investigated over time using a pH dependent live cell stain and automated microscopy. Furthermore, the impact of CD47, galectin-3 or galectin-9 antibody-mediated blockade on phagocytosis of PDAC cells was evaluated.Results The immune checkpoint molecules CD47 as well as galectin-3 and -9 were heterogeneously expressed by PDAC cells. Overall, expression of galectins was higher in macrophages compared to tumor cells and differed tremendously between M1- and M2-like macrophages. Phagocytosis of tumor cells by M2-like macrophages varied depending on the PDAC cell line, while M1-like macrophages exhibited low to no phagocytotic potential. Furthermore, antibody-mediated blockade of CD47 significantly increased phagocytosis of tumor cells by M2-like but not M1-like macrophages, while blocking of galectins did not significantly impact phagocytosis rates.Conclusion Although CD47 blockade significantly increased phagocytosis, many PDAC cells remained untargeted by M2-like macrophages and blocking of galectins did not impact M2-like macrophage mediated phagocytosis of PDAC cells, indicating that blocking of CD47 or galectins alone is not sufficient to induce elimination of PDAC cells. These findings suggest that regulation of phagocytosis is not depended on CD47 exclusively, thus multiple factors may need to be targeted simultaneously. As galectin-3 has been described to cooperate with CD47 in suppressing phagocytosis in other entities, combination of both strategies should be further investigated.