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Extracellular vesicles (EVs) play a modulatory role in cancer communication and progression, and interestingly, several tumor\u2010associated 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\u2013protein interaction and pathway analysis reveal individual signatures on the EVs released by 2D\u2010 and 3D\u2010cultured 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\u2010EV cargo and its biological role.<\/jats:p>","DOI":"10.1002\/advs.202300588","type":"journal-article","created":{"date-parts":[[2023,6,21]],"date-time":"2023-06-21T00:48:29Z","timestamp":1687308509000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Differential Protein and Glycan Packaging into Extracellular Vesicles in Response to 3D Gastric Cancer Cellular Organization"],"prefix":"10.1002","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0266-9093","authenticated-orcid":false,"given":"\u00c1lvaro M.","family":"Martins","sequence":"first","affiliation":[{"name":"i3S\u2010Institute for Research and Innovation in Health University of Porto  Rua Alfredo Allen 208 Porto 4200-135 Portugal"},{"name":"IPATIMUP\u2010Institute of Molecular Pathology and Immunology University of Porto  Rua J\u00falio Amaral de Carvalho 45 Porto 4200-135 Portugal"},{"name":"Instituto de Ci\u00eancias Biom\u00e9dicas Abel Salazar (ICBAS) University of Porto  R. Jorge de Viterbo Ferreira Porto 4050-313 Portugal"}]},{"given":"T\u00e2nia M.","family":"Lopes","sequence":"additional","affiliation":[{"name":"i3S\u2010Institute for Research and Innovation in Health University of Porto  Rua Alfredo Allen 208 Porto 4200-135 Portugal"},{"name":"IPATIMUP\u2010Institute of Molecular Pathology and Immunology University of Porto  Rua J\u00falio Amaral de Carvalho 45 Porto 4200-135 Portugal"}]},{"given":"Francisca","family":"Diniz","sequence":"additional","affiliation":[{"name":"i3S\u2010Institute for Research and Innovation in Health University of Porto  Rua Alfredo Allen 208 Porto 4200-135 Portugal"},{"name":"IPATIMUP\u2010Institute of Molecular Pathology and Immunology University of Porto  Rua J\u00falio Amaral de Carvalho 45 Porto 4200-135 Portugal"},{"name":"Instituto de Ci\u00eancias Biom\u00e9dicas Abel Salazar (ICBAS) University of Porto  R. Jorge de Viterbo Ferreira Porto 4050-313 Portugal"}]},{"given":"Jos\u00e9","family":"Pires","sequence":"additional","affiliation":[{"name":"i3S\u2010Institute for Research and Innovation in Health University of Porto  Rua Alfredo Allen 208 Porto 4200-135 Portugal"},{"name":"IPATIMUP\u2010Institute of Molecular Pathology and Immunology University of Porto  Rua J\u00falio Amaral de Carvalho 45 Porto 4200-135 Portugal"},{"name":"Faculty of Medicine of the University of Porto  Alameda Prof. Hern\u00e2ni Monteiro Porto 4200-319 Portugal"}]},{"given":"Hugo","family":"Os\u00f3rio","sequence":"additional","affiliation":[{"name":"i3S\u2010Institute for Research and Innovation in Health University of Porto  Rua Alfredo Allen 208 Porto 4200-135 Portugal"},{"name":"IPATIMUP\u2010Institute of Molecular Pathology and Immunology University of Porto  Rua J\u00falio Amaral de Carvalho 45 Porto 4200-135 Portugal"},{"name":"Faculty of Medicine of the University of Porto  Alameda Prof. Hern\u00e2ni Monteiro Porto 4200-319 Portugal"}]},{"given":"Filipe","family":"Pinto","sequence":"additional","affiliation":[{"name":"i3S\u2010Institute for Research and Innovation in Health University of Porto  Rua Alfredo Allen 208 Porto 4200-135 Portugal"},{"name":"IPATIMUP\u2010Institute of Molecular Pathology and Immunology University of Porto  Rua J\u00falio Amaral de Carvalho 45 Porto 4200-135 Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7606-1120","authenticated-orcid":false,"given":"Daniela","family":"Freitas","sequence":"additional","affiliation":[{"name":"i3S\u2010Institute for Research and Innovation in Health University of Porto  Rua Alfredo Allen 208 Porto 4200-135 Portugal"},{"name":"IPATIMUP\u2010Institute of Molecular Pathology and Immunology University of Porto  Rua J\u00falio Amaral de Carvalho 45 Porto 4200-135 Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0286-6639","authenticated-orcid":false,"given":"Celso A.","family":"Reis","sequence":"additional","affiliation":[{"name":"i3S\u2010Institute for Research and Innovation in Health University of Porto  Rua Alfredo Allen 208 Porto 4200-135 Portugal"},{"name":"IPATIMUP\u2010Institute of Molecular Pathology and Immunology University of Porto  Rua J\u00falio Amaral de Carvalho 45 Porto 4200-135 Portugal"},{"name":"Instituto de Ci\u00eancias Biom\u00e9dicas Abel Salazar (ICBAS) University of Porto  R. 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