Cancer cell glycocalyx thickness is a druggable physical phenotype for improving cellular immunotherapy

The abnormally thick glycocalyx of cancer cells can physically limit immune-cell engagement and reduce the efficacy of cellular immunotherapies, but strategies for identifying pharmacological agents that relieve this barrier remain limited. Here, we establish glycocalyx thickness as a pharmacologically actionable physical phenotype. Disruption of mucin-type O-glycosylation reduced glycocalyx thickness and increased NK- and CAR-NK-mediated killing, demonstrating that pharmacological glycocalyx remodeling can enhance innate and CAR-directed cytotoxicity. To screen this phenotype directly, we developed a scanning angle interference microscopy (SAIM) platform using complementary leucine zipper pairs as a stable extracellular plasma-membrane reference. A focused screen of glycosylation and metabolic inhibitors resolved compound- and cell-type-specific nanoscale changes in glycocalyx thickness and identified several thinning agents. Castanospermine, an inhibitor of early N-glycan processing, was among the strongest hits and reduced mucin-probe binding in an independent mucin-rich gastric cancer model. Castanospermine-treated gastric cancer cells retained MUC17 antibody recognition and showed increased physical avidity for MUC17 CAR-T cells, while castanospermine enhanced MUC17 CAR-T-mediated tumor control in real-time co-culture assays. These findings establish direct glycocalyx-thickness measurement as a physical-phenotype-guided screening strategy and identify castanospermine as a candidate modulator for combination with MUC17 CAR-T cells.