In this study, we revealed a novel molecule called Siglec-9 that acts as an immune checkpoint on macrophages in GBM, significantly impacting the tumor immune environment and immunotherapy response. This research provides new clues to address the above-mentioned challenges.

We performed single-cell RNA sequencing and spatial transcriptomic analysis on samples from 24 GBM patients, including primary tumors, recurrent tumors, and patients who received anti-PD-1 antibody as neoadjuvant therapy. We found that in patients who showed ineffective responses to anti-PD-1 therapy, the tumor-associated macrophages (TAMs) with high expression of the Siglec-9 gene were predominant. Furthermore, we discovered that although anti-PD-1 therapy could alter the tumor immune environment in some GBM patients, activating T cells and increasing the expression of certain chemokine and interferon-γ-related genes, it could not overcome the overall immune suppression state of the tumor microenvironment, particularly the immune inhibitory function of Siglec-9 high-expressing macrophages (Siglec-9⁺ TAMs).

We further identified two distinct subpopulations of Siglec-9⁺ TAMs: Siglec-9⁺ MARCO⁺ TAMs and Siglec-9⁺SEPP1SIGLEC9⁺ TAMs. Spatial transcriptomic analysis revealed that these two TAM subpopulations originated from CD14⁺ monocytes that migrated stepwise into the tumor and underwent differentiation. Siglec-9⁺MARCO⁺ TAMs possibly represented a transitional state where circulating monocytes differentiated into Siglec-9⁺SEPP1⁺ TAMs. Notably, cross-comparison with published GBM single-cell sequencing data showed that Siglec-9⁺ TAMs resembled macrophages co-expressing GPNMB/CSTB/MRC1 in GBM patients treated with immune checkpoint blockade (ICB), while they were not enriched in the dataset of recurrent GBM patients (Lee et al., Nat Commun 2021; Antunes et al., Nat Neurosci 2021). Thus, anti-PD-1 therapy might reprogram Siglec-9⁺ TAMs. This study provides insights into the reasons behind the response and non-response effects produced by neoadjuvant anti-PD-1 therapy.

Siglec-9 belongs to the Siglec family, which consists of immunoglobulin-like lectins expressed on the surface of macrophages and other myeloid immune cells (Duan et al., Annu Rev Immunol 2020). These receptors act as immune checkpoint molecules by delivering immune inhibitory signals when they bind to glycoproteins containing sialic acid residues on the host/cancer cell membrane. Through studies using a mouse model with SiglecE (the murine functional homolog of human Siglec-9) gene knockout in intracranial tumor models, we found that the deletion of SiglecE could inhibit GBM tumor growth, alter the immune-suppressive phenotype of macrophages to a more anti-tumor phenotype, and significantly enhance T cell activation and proliferation. Moreover, we conducted a series of in vivo and in vitro experiments utilizing mouse models and clinical samples from human patients to further validate the role of Siglec-9⁺/SiglecE⁺ TAMs in inhibiting tumor growth in Siglece knockout mice. We observed that Siglec-9⁺/SiglecE⁺ TAMs directly interact with T cells. Additionally, to gain further insights into the clinical implications of targeting Siglec-9, we evaluated the tutor growth of Siglece knockout mice  upon receiving anti-PD-1 therapy and found significantly prolonged survival. Furthermore, we found the  competitive protein that specifically targeted Siglec-9, could substantially extended the survival of mice bering glioblastoma.

Figure 1: Schematic Illustration of Macrophage Specialization and T Cell Interaction in Human GBM and Mouse Models | Recurrent GBM tumors are accompanied by increased angiogenesis, loss of microglial cells, and infiltration of TAMs derived from monocytes. After neoadjuvant anti-PD-1 therapy, especially in non-responders, Siglec-9⁺ TAMs accumulated. In a mouse GBM model, targeting Siglec-E (mouse homolog of Siglec-9) on macrophages can preferentially activate T cells by secreting chemokines, interacting with co-stimulatory factors, and upregulating the IFN-γ-related pathway, thereby enhancing the effectiveness of anti-PD-1/PD-L1 therapy. Siglec-9/Siglec-E represents an immune checkpoint on macrophages and serves as a potential target for GBM immunotherapy.

This study provides new insights for the treatment of glioblastoma, suggesting that Siglec-9 is a novel macrophage immune checkpoint molecule that can be targeted to enhance the efficacy of anti-PD-1/PD-L1 therapy for GBM. The study also demonstrates that combining single-cell analysis of human clinical samples with translational research in mouse models can effectively identify novel immune checkpoints (Figure 1).