GlycoSS: A DNA Glycosignal\nSieve for Deciphering Spatially\nResolved EpCAM-Specific Glycoforms

Malignant transformation of cancer is often accompanied\nby aberrant\nglycopatterns. Epithelial–mesenchymal transition (EMT) is a\ncrucial biological process in cancer migration and invasion, accelerating\ncancer deterioration. High-precision analysis of protein–glycan\nspatial profiling in the EMT process is essential for elucidating\nglycosylation functions and cancer progression. However, the diversity\nof glycans in composition and conformation complicates their spatial\nanalysis. Here, we develop a DNA glycosignal sieve (GlycoSS) visualization\nplatform for screening glycoform expression with a protein spatial\ndimension. GlycoSS utilizes protein-anchored DNA nanoscanners of distinct\nlengths to control glycosignal readout, enabling protein–glycan\ndistance modulations, and simultaneously orthogonally amplify glycoform\noutput through signal amplification by an exchange reaction. Using\nGlycoSS, we screened EpCAM-specific hypoglycosylated glycoform signals\nin different breast cancer cell subtypes, especially characterizing\nthe spatial distribution of glycans on the MCF-7 cell surface. Considering\nthat the EpCAM-specific <i>N</i>-glycan dysregulation in\nEMT is pivotal, GlycoSS revealed dynamic glycan fluctuations during\nIGF-1-induced EMT, revealing that the <i>N</i>-glycans were\npositively associated with tumor malignancy and metastasis. GlycoSS\nis anticipated to accelerate the identification of aberrant <i>N</i>-glycosylation in tumor progression, advancing systemic\nglycobiology insights. Notably, GlycoSS is capable of analyzing diverse\nglycoprotein profiles, offering additional dimensions into the role\nof glycoprotein nanoenvironments in regulating membrane protein function.