Differential Protein and Glycan Packaging into Extracellular Vesicles in Response to 3D Gastric Cancer Cellular Organization

Abstract Alterations of the glycosylation machinery are common events in cancer, leading to the synthesis of aberrant glycan structures by tumor cells. Extracellular vesicles (EVs) play a modulatory role in cancer communication and progression, and interestingly, several tumor‐associated glycans have already been identified in cancer EVs. Nevertheless, the impact of 3D tumor architecture in the selective packaging of cellular glycans into EVs has never been addressed. In this work, the capacity of gastric cancer cell lines with differential glycosylation is evaluated in producing and releasing EVs when cultured under conventional 2D monolayer or in 3D culture conditions. Furthermore, the proteomic content is identified and specific glycans are studied in the EVs produced by these cells, upon differential spatial organization. Here, it is observed that although the proteome of the analyzed EVs is mostly conserved, an EV differential packaging of specific proteins and glycans is found. In addition, protein–protein interaction and pathway analysis reveal individual signatures on the EVs released by 2D‐ and 3D‐cultured cells, suggesting distinct biological functions. These protein signatures also show a correlation with clinical data. Overall, this data highlight the importance of tumor cellular architecture when assessing the cancer‐EV cargo and its biological role.