Scheme 1. Working Principle of the FLAG Strategy a a This FLAG strategy offers visualization of PD-L1-specific glycosylation on tissue sections through FRET between the PD-L1 aptamer (MJ5C)-labeled FAM and a lectin (Con A)-conjugated Cy3.

Exosomal programmed cell death ligand 1 (exoPD‐L1) has emerged as a promising biomarker for cancer diagnosis and immunotherapy outcome prediction. However, the existing quantitation methods are incapable of addressing the heterogeneity of exoPD‐L1 glycosylation, which has been demonstrated to be the institutional basis for PD‐L1/PD‐1 interaction and the crucial participant in inhibiting the activity of CD8+ T cells. Herein, an aptamer‐ and lectin‐induced proximity ligation assay combined with quantitative real‐time polymerase chain reaction for precise quantitation of glycosylated exoPD‐L1 is developed. Leveraging the metabolism‐free lectin labeling of glycosylation, the glycosylation‐independent aptamer tagging of PD‐L1, and excellent selectivity of dual‐recognition, this method enables glycosylated exoPD‐L1 quantitation with high sensitivity and selectivity in a wash‐free manner. As a result, this method is able to distinguish the levels of glycosylated exoPD‐L1 between healthy donors and cancer patients with sensitivity and specificity of 100%. Compared with the total circulating exoPD‐L1 level, glycosylated exoPD‐L1 is for the first time identified to be a more reliable biomarker for tumor diagnosis. Overall, this strategy holds a great potential for revealing the significance of exoPD‐L1 glycosylation and converting glycosylated exoPD‐L1 into a reliable clinical indicator.

Exosomal glycoproteins play significant roles in many physiological and pathological procedures. However, the current methods for studying exosomal glycoproteins have low sensitivity or can affect exosomal biological function. Herein, we developed a proximity dual-tagging strategy using an induced hybridization chain reaction (HCR) from the target's non-functional epitope for amplified visualization and functional exploration of exosomal protein-specific glycosylation. This strategy leverages dualtagging based on the aptamer with little influence on target function and metabolic glycan labelling, and the rigid product and high sensitivity of HCR. The method improves the signal of visualizing exosomal PD-L1 (exoPD-L1) by 7.7-fold compared with the signal without HCR amplification without affecting the natural exoPD-L1/PD-1 interaction. As a result, we verified that the interaction between exoPD-L1 and PD-1 positive cells is positively correlated to the glycosylation level of exoPD-L1. Overall, we have developed a sensitive method with little functional influence to visualize exosomal protein-specific glycosylation in situ, offering a powerful tool for studying the biological implications of exosomal glycoproteins.

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