Sialylated Glycoproteins-Siglec Pathway: A New Target for Prostate Cancer Immunotherapy
On October 22, 2024, the team of James D. Brooks from Stanford University published an article entitled "Sialylated glycoproteins suppress immune cell killing by binding to Siglec-7 and Siglec-9 in prostate cancer" in The Journal of Clinical Investigation. This article focuses on the key role of glycosylation in the immune escape of Prostate Cancer (PCa) and proposes a new immunotherapy strategy targeting Siglec-7/9.
Although immune checkpoint inhibitors (ICIs) have been successfully used in the treatment of various tumors, their efficacy in prostate cancer is limited, and only a very small number (about 1%) of patients with mismatch repair deficiency can benefit from them. Siglecs (sialic acid-binding immunoglobulin-like lectins), as an immune regulatory receptor that recognizes Sialic Acid, are gradually gaining attention in tumor immunosuppression. The study found that Siglec-7 and Siglec-9 are highly expressed in various stages of prostate cancer, especially on immune cells in the tumor microenvironment. These Siglec molecules inhibit the activity of immune cells by recognizing sialylated glycans chains on the surface of tumor cells, thereby helping tumors escape the attack of the immune system.
This paper first studied the expression of Siglec-7 and Siglec-9 in PCa tissues. Analysis of the two major prostate cancer databases TCGA-PRAD and MSKCC showed that the mRNA levels of Siglec-7 and Siglec-9 in tumor tissues were significantly higher than those in normal tissues. Not only that, the expression level was positively correlated with higher tumor grade (Gleason score) and worse recurrence-free survival (RFS). To further determine the source of their expression, the authors combined the CIBERSORTx algorithm with scRNA-seq analysis and found that these two receptors were mainly expressed in myeloid immune cells, especially tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs).
To further elucidate the Siglec spectrum of PCa, the authors analyzed Siglec expression in PCa tumor tissues at three different stages (primary, hormone-sensitive, and castration-resistant) by scRNA-seq, and found that Siglec-7 and Siglec-9 were expressed in myeloid immune cells at all stages of PCa. In addition, Siglec-10 was also highly expressed. Subsequently, immunofluorescence and confocal microscopy techniques were used to demonstrate that Siglec-7 and Siglec-9 were highly expressed on macrophages in PCa bone metastasis tissues.
The authors explored the presence of sialylated glycans on the surface of PCa cells. Sialic acid fluorescence assays revealed that sialylated glycans were present on the surface of all tested PCa cell lines. SNA and MAL-II lectin staining revealed that sialylated glycans on the surface of PCa cells were mainly composed of α-2,6 sialic acid. Glycoproteomic Analysis showed that more than 85% of the differential glycosylation between PCa tumor tissue and normal prostate came from increased sialylation, and further GO analysis showed that these differential glycoproteins were enriched in pathways related to immune response/antigen presentation, metabolism, cell adhesion and communication. Gene expression analysis of sialylated glycans biosynthesis in tumor tissues of PCa patients showed that α-2,3 and α-2,6 sialyltransferases showed significant changes in PCa tissues compared with adjacent normal tissues.
To further determine whether Siglec-7 and Siglec-9 ligands are present on the surface of PCa cells, the authors performed direct staining using recombinant chimeras of the binding regions of Siglec-7 or Siglec-9 molecules fused to the human IgG1 Fc domain, referred to as Siglec-7-Fc or Siglec-9-Fc. The results showed the presence of Siglec-7 and Siglec-9 ligands on the surface of PCa cells. In addition, all PCa cells exhibited higher levels of Siglec-9-Fc binding compared to Siglec-7-Fc binding. Subsequently, flow cytometry and immunofluorescence experiments determined that removal of sialic acid from the surface of PCa cells with sialidase resulted in a significant reduction in the binding of Siglec-7-Fc and Siglec-9-Fc. Finally, the expression of Siglec-7 and Siglec-7 ligands in tumor tissues from PCa patients was detected by immunohistochemistry and flow cytometry. The results showed that the expression levels of Siglec-7 and Siglec-9 ligands in tumor tissues were significantly increased compared with adjacent normal tissues.
To further verify the role of Siglec-7 and Siglec-9 in immune escape, the authors constructed a Humanized Mouse Model: after the injection of PC3 or 22Rv1 cells to establish xenograft tumors, human PBMCs and CD8+ T cells were injected on the 7th day, and Siglec-7/9 antibodies or IgG controls were injected on the 13th and 19th days. The results showed that the tumor volume and weight of the treatment group were significantly reduced, and T cell infiltration in the tumor was significantly enhanced, apoptosis increased, proliferation decreased, and vascularization increased. In addition, if human PBMCs were not injected, anti-Siglec antibodies could not inhibit tumor growth, indicating that their efficacy depends on immune cells.
Through large-scale CRISPRi screening (209,070 sgRNAs) + LC-MS/MS mass spectrometry immunoprecipitation dual strategy, the authors finally locked a key ligand - CD59. After Knocking Out CD59, the binding ability of Siglec-9-Fc to PC3 cells decreased significantly. At the same time, the killing ability of CD8+ T cells on CD59 knockout cells was significantly enhanced, indicating that CD59 plays a substantial role in immunosuppression.
Fig. 1 CD59 is a candidate ligand for Siglec-9 in PCa. (Wen, et al., 2024)
In summary, this study revealed the immunomodulatory role of Siglec-7 and Siglec-9 in prostate cancer and confirmed their potential as new immune checkpoints. By targeting these molecules, the immune system's ability to attack tumors can be enhanced, providing new ideas and potential therapeutic targets for immunotherapy of prostate cancer.
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