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The success of malignant tumors is conditioned by the intercellular communication between tumor cells and their microenvironment, with extracellular vesicles (EVs) acting as main mediators. While the value of 3D conditions to study tumor cells is well established, the impact of cellular architecture on EV content and function is not investigated yet. Here, a recently developed 3D cell culture microwell array is adapted for EV production and a comprehensive comparative analysis of biochemical features, RNA and proteomic profiles of EVs secreted by 2D vs 3D cultures of gastric cancer cells, is performed. 3D cultures are significantly more efficient in producing EVs than 2D cultures. Global upregulation of microRNAs and downregulation of proteins in 3D are observed, indicating their dynamic coregulation in response to cellular architecture, with the ADP-ribosylation factor 6 signaling pathway significantly downregulated in 3D EVs. The data strengthen the biological relevance of cellular architecture for production and cargo of EVs. S.R. and J.C. contributed equally to this work as co-first authors. C.O. and I.N. contributed equally to this work as co-senior authors. The authors thank Celso Reis and Joy Burchell for having generously shared the anti-Mucin-1 antibody, Gabriela Almeida and Mafalda Santos for sharing the H&E images from MKN45 and MKN74 tumors in mice, Maria Lazaro for the technical support with imaging flow cytometry data, Tanja Gainey-Schleicher and Elena Grueso Navarro for the support with EV–cell association assay, and Deborah Lawrie-Blum for proofreading the manuscript. S.R., R.K., J.C., C.O., A.T., A.K., and I.N. were involved in the conception and design of the study; S.R., J.C., and P.O. were involved in the establishment and characterization of 3D microwell array culture of GC cells, characterization of EVs, small RNA sequencing experiments, data analysis and validation, and functional assays; D.S., M.V., N.W., and R.K. were involved in all proteomics studies including data analysis and validation, and the analysis of cell–EV association; S.R., J.C., P.O., C.O., D.S., A.K., J.-C.S., A.T., and I.N. analyzed the data; S.R., J.C., P.O., C.O., A.K., and I.N. wrote the manuscript; S.R., J.C., P.O., C.O., A.K., and I.N. critically reviewed the manuscript; and all authors gave final approval of the manuscript.

Pancreatic beta-cells are responsible for insulin secretion that regulates blood glucose homeostasis. In the development of type II diabetes, a progressive impairment of insulin secretion by the pancreatic beta-cells occurs called beta-cell dysfunction or beta-cell failure. Chronic hyperglycemia has been shown being involved in beta-cell dysfunction, a phenomenon known as glucotoxicity. The molecular mechanisms underlying the impairment of insulin secretion by beta-cells induced by glucotoxicity are still not fully understood. In this work, quantitative proteomics was employed to identify early key players involved in beta-cell dysfunction induced by glucotoxicity. For this, the stable isotope labeling by amino acids in cell culture strategy was used on the slowly-growing rat beta-cell line INS-1E. We showed that the stable isotope labeling by amino acids in cell culture approach did not induce any detectable biological effects on these beta-cells, as measured at both the transcriptomic and proteomic levels. Proteins differentially expressed between control cells and cells submitted to chronic high glucose concentrations were identified and verified. The results obtained reinforce the link between glucotoxicity and lipogenesis and suggest that the fatty acid metabolism pathway may rapidly be stimulated in beta-cells submitted to chronic high glucose concentrations.