Siglec Ligand Immunohistochemistry Reveals Association With Immune Exclusion and Survival

Sialic acids are overexpressed in many cancers, and binding of sialic acid via sialic acid binding immunoglobulin-like lectins (Siglecs) may contribute significantly to immune evasion and cancer progression. This important resistance mechanism in the tumor immune microenvironment has been understudied, partially due to the lack of useful reagents. Here, we developed and optimized an immunohistochemistry staining protocol for novel reagents that detect 3 types of Siglec-engaging sialoglycans (HYDRA-3, -7, and -9, which detect sialoglycans recognized by Siglec-3, -7, and -9, respectively) in the tumor immune microenvironment. We evaluated HYDRA staining across 10 different cancer types across whole slides, finding that HYDRA-9 exhibited the highest overall staining range, with HYDRA-3 and -7 showing lower to moderate staining across all tested tumor types. To correlate HYDRA staining patterns and immune infiltration in melanoma, we stained melanoma tissue microarrays with the 3 HYDRA reagents and compared HYDRA staining profiles with a 6-plex multiplex immunofluorescence panel targeting CD8, CD163, FoxP3, PD1, PDL1, and Sox10/S100. Siglec-3 and -9 sialoglycan ligand expression negatively correlated with CD8 T cell infiltration (r = -0.28/P = .002 and r = -0.29/P = .001, respectively), particularly at the tumor-stromal interface (r = -0.37/P < .001 and r = -0.44/P < .001, respectively). Additionally, a high ratio of Siglec-3 and -9 ligand expression at the tumor-stromal interface versus the tumor core was associated with reduced overall survival (Hazard's ratio: 2.60 and 2.11, respectively), whereas CD8 infiltration was not associated with survival outcomes in our cohort. Taken together, the expression levels and spatial distribution of Siglec-engaging sialoglycans may play a role in patient prognosis, potentially representing a biomarker of survival that is independent of conventional metrics of an inflamed tumor microenvironment. This study highlights the need for further investigation of Siglec ligand expression as a predictive and prognostic biomarker of treatment response and resistance.