Novel siglec probes identify aberrant sialic acid expression linked to immune exclusion and survival in melanoma

Summary: Abstract Body: Sialoglycans display aberrant overexpression in a range of cancers, where their interaction with sialic acid-binding immunoglobulin-like lectins (Siglecs) may play a role in facilitating immune evasion and tumor progression. However, Siglec implicated mechanisms of evasion and progression within the tumor immune microenvironment (TIME) remain underexplored, partially due to the limited availability of high-affinity detection probes. In this study, we developed and optimized an immunohistochemistry protocol using novel reagents—HYDRA-3, -7, and -9—to detect Siglec-3, -7, and -9 sialoglycan ligands, respectively. To correlate HYDRA staining patterns with immune infiltration, we analyzed melanoma tissue microarrays (TMAs) constructed using tumor core and tumor-stromal boundary samples from 135 patients with known 5-year survival outcomes. TMAs stained with the three HYDRA reagents were directly compared to TMAs stained with a 6-plex immunofluorescence panel targeting CD8, CD163, FoxP3, PD1, PDL1, and Sox10/S100. Notably, Siglec-3 and Siglec-9 ligand expression negatively correlated with CD8 T-cell infiltration (r = -0.28, p = 0.002 and r = -0.29, p = 0.001, respectively), particularly at the tumor-stromal interface (r = -0.37, p < 0.001 and r = -0.44, p < 0.001, respectively). Moreover, a high ratio of Siglec-3 and Siglec-9 ligand expression at the tumor-stromal interface relative to the tumor core was associated with reduced overall survival (HR = 2.60, 95% CI 1.31-5.18 and HR = 2.11, 95% CI 1.01-4.41, respectively). These findings suggest that the spatial distribution of Siglec-engaging sialoglycans, rather than total expression, plays a key role in shaping the TIME and influencing patient outcomes, possibly as a result of abnormal sialoglycan overexpression along the tumor-stromal interface promoting an immune excluded environment. Jeremy Ellis¹, Elizabeth Will¹, Aleksandra Ogurtsova¹, Logan Engle¹, Janis Taube¹, Joel Sunshine¹ 1. Johns Hopkins University, Baltimore, MD, United States. Pigmentation, Melanoma, and Melanoma Immune Surveillance