3D Cellular Architecture Affects MicroRNA and Protein Cargo of Extracellular Vesicles

Abstract

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.

3D cell culture stimulates the secretion of in vivo like extracellular vesicles

“…However, their parent cells cultured between 2D and 3D systems exhibited similar miRNA expression profile. Rocha, et al 56 also observed that striking differences between small RNAs detected in EVs and in donor cells, which support our results and other research reports indicating a specific sorting process of small RNAs into EVs 26,27 . As anticipated, replicates (2D-cell miRNAs and 3Dcell miRNAs) were clustered together.…”

“…The harvested EVs showed typical round, cup shape via SEM imaging in both 2D and 3D culture conditions (Figure 2 B and D), while 3D culture-derived EVs showed the more dense distribution of smaller size. These observations were in agreement with Rocha, et al that exosomes released by tissue-engineered tumors were smaller than exosomes released by the same cells cultured in monolayer, and the 3D system is more active for producing exosomes 56,57 . It is interesting that our observation is the same as Rocha, et al report that the maximum period for conditioned media collection was 48 h for 2D.…”

“…It is interesting that our observation is the same as Rocha, et al report that the maximum period for conditioned media collection was 48 h for 2D. Rocha, et al also observed that in 3D this period could be extended for another 4 d without compromising cell viability 56 . We characterized the confluency property for both 2D and 3D cultures (see Figure s2).…”

“…As anticipated, replicates (2D-cell miRNAs and 3Dcell miRNAs) were clustered together. It is interesting that we observed the significant difference of miRNAs expression profile between 2D and 3D culture derived EVs, while Rocha, et al showed high similarity 56 . The experimental data explaining this phenomenon are still scarce, and more investigation should be performed for a definitive conclusion.…”

Exosomes in osteoarthritis and cartilage injury: advanced development and potential therapeutic strategies

“…Some studies have reported that exosome produced by 3D cultures (Exos/EVs-3D) has more advantages than exosome produced by 2D cultures (Exos/EVs-2D) [50][51][52][53][54][55][56][57] in terms of the size, content, function and production efficiency of exosomes. The functions of exosomes differ according to their content, while Exos/EVs-3D contain a different profile of proteins and genetic materials compared to Exos/EVs-2D [52] .…”