Multi-modal spatial glycomic analysis of lymphoid aggregates in prostate and pancreatic cancer tissues

Altered N-glycosylation plays a critical role in immune regulation, yet the spatial distribution of N-glycans within immune niches of immunologically "cold" tumors remain poorly defined. Here, we combine matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) with highly multiplexed immunohistochemistry using photocleavable mass tags. This integrated workflow enables spatial mapping of N-glycosylation and immune markers within lymphoid aggregates (LAs) in prostate and pancreatic ductal adenocarcinoma tissues, two malignancies that respond poorly to immunotherapy. Given the role of N-glycosylation in immune regulation, spatially resolved N-glycan profiling may provide insight into molecular heterogeneity within lymphoid aggregates and their surrounding tumor microenvironment.

To assess the relationship between N_glycosylation and immune markers, candidate LAs from tissue samples collected from patients with varying stages of prostate cancer, PCa (n=49) and pancreatic ductal adenocarcinoma, PDAC (n=26) were identified via histological stains prior to enzymatic release of N-glycans and MALDI-MSI. MALDI-IHC was used to target immune cell markers and extracellular matrix proteins.

In tissues with LAs, three N-glycan structural classes were identified when detectable: oligo-mannose, fucosylated tetra-antennary, and bisecting N-acetylglucosamine in fucosylated bi,- tri- and tetra- antennary structures, with some N-glycans common to both tumor types. LAs showed predominantly central CD20 and CD44 signal surrounded by CD3 signal localized along the tumor periphery.

Our analysis revealed unique N-glycan patterns within and surrounding LAs, with variable profiles in distant regions, highlighting heterogeneity in LA-associated N-glycan profiles.