GlycoAvatars: Bead-Coated Membrane Models for Studying the Cancer-Immune Cells Interactome

Immature O -glycosylation of plasma membrane proteins, characterized by the presence of glycosites with a single GalNAc residue (the Tn antigen) rather than more extended and complex glycans, is a prominent feature of many advanced solid tumors across different origins and aetiologies. This form of glycosylation has been strongly associated with poor prognosis and is known to play a crucial role in the progression of the disease. Notably, it has been directly implicated in the crosstalk with the immune system, inducing tolerogenic phenotypes in both innate and adaptive immune cells. Despite this, the specific receptors involved in these interactions remain largely unidentified. To address this gap, we propose a novel high-throughput proteomics assisted strategy for characterizing the glycan interactome using plasma membrane-derived glycoprotein coated magnetic beads. Glycoproteins were isolated from plasma membranes of glycoengineered cell models with immature glycosylation and were then cloaked on the surface of magnetic beads, creating simplified cell “GlycoAvatars.” These GlycoAvatars were primarily employed to characterize the interactome between gastric and colorectal cancer cell lines and immune cells (dendritic cells and macrophages), providing a tool for identifying potentially relevant molecular nodes in glycan-mediated immune synapses, foreseeing their application in deciphering tumor-immune interactions.