Role of sialic acid-siglec axis in pancreatic cancer immune evasion

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy. A key factor contributing to the lethality of PDAC is immune evasion. Tumor cells suppress anti-tumor immunity by activating immune checkpoint proteins on immune cells including macrophages. Siglec receptors are one of the main checkpoint molecules on macrophages. The ligand for Siglecs is sialic acid. To effectuate immune suppression via Siglecs, PDAC cells increase their surface sialylation by upregulating the expression of sialyltransferases such as ST6GAL1. ST6GAL1 adds an alpha2,6-linked sialic acid to N-glycans. Both alpha2,6 sialylation and ST6GAL1 expression are markedly increased in PDAC cells. Our prior studies revealed potent tumor-autonomous functions for ST6GAL1. Our new unpublished results suggest that the tumorigenic effects of ST6GAL1 are also driven by its role in creating sialoglycan ligands for macrophage Siglecs. Macrophages that are polarized to an immunosuppressive M2 phenotype are key contributors to PDAC progression. The goal of our study is to determine whether macrophage Siglecs are promising therapeutic targets for preventing M2 polarization, thus restoring anti-tumor immunity and preventing PDAC progression. We investigated macrophage polarization in our genetically engineered mouse models of PDAC, utilizing either pancreas-specific knock-in of oncogenic K-ras (KC mouse) or K-ras in combination with ST6GAL1 knock-in (KSC mouse). Single-cell RNA sequencing, flow cytometry, and immunohistochemistry showed an increased number of M2 macrophages in KSC vs. KC pancreata. Macrophages co-cultured with ST6GAL1-overexpressed PDAC cells showed increased expression of M2 markers. But adding Siglec-blocking antibodies reversed the ST6GAL1-mediated M2 polarization, indicating that the ST6GAL-mediated M2 polarization of macrophages is mediated through Siglec signaling. Additionally, we determined the phagocytotic capacity of macrophages, which is an established functional readout for anti-tumor behavior of macrophages. Immunofluorescence microscopy and flow cytometry showed that the phagocytotic capability of macrophages was suppressed by ST6GAL1-overexpressed PDAC cells but recovered by Siglec blocking antibody. Overall, our data demonstrate that alpha2,6 sialic acids on PDAC cells engage with Siglecs on macrophages to induce polarization of macrophages into an immunosuppressive M2 phenotype. Collectively, these results reveal a potential mechanism for targeting Siglecs as a promising immune checkpoint therapy for PDAC patients with high levels of ST6GAL1. Traditional T cell-based checkpoint therapies have limited effectiveness in PDAC. Targeting Siglec checkpoints presents an opportunity to enhance the anti-tumor activity of macrophages, the primary immune cell type within the PDAC tumor microenvironment.