Leucine zipper-based SAIM imaging identifies therapeutic agents to disrupt the cancer cell glycocalyx for enhanced immunotherapy

Park, S.; Paek, J. H.; Colville, M. J.; Huang, L.-T.; Struzyk, A. P.; Womack, S. J.; Neelamegham, S.; Reesink, H. L.; Paszek, M. J.

doi:10.1101/2024.12.05.627089

bioRxiv · 10.1101/2024.12.05.627089

Leucine zipper-based SAIM imaging identifies therapeutic agents to disrupt the cancer cell glycocalyx for enhanced immunotherapy

Park, S.·Paek, J. H.·Colville, M. J.·Huang, L.-T.·Struzyk, A. P.·Womack, S. J.·Neelamegham, S.·Reesink, H. L.·Paszek, M. J.

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Abstract

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.

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Park, S., Paek, J. H., Colville, M. J., Huang, L.-T., Struzyk, A. P., Womack, S. J., Neelamegham, S., Reesink, H. L., Paszek, M. J.. 2024-12-06. Leucine zipper-based SAIM imaging identifies therapeutic agents to disrupt the cancer cell glycocalyx for enhanced immunotherapy. https://doi.org/10.1101/2024.12.05.627089

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