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The improvement of culturing techniques to model the environment and physiological conditions surrounding tumors has also been applied to the study of extracellular vesicles (EVs) in cancer research. EVs role is not only limited to cell-to-cell communication in tumor physiology, they are also a promising source of biomarkers, and a tool to deliver drugs and induce antitumoral activity. In the present review, we have addressed the improvements achieved by using 3D culture models to evaluate the role of EVs in tumor progression and the potential applications of EVs in diagnostics and therapeutics. The most employed assays are gel-based spheroids, often utilized to examine the cell invasion rate and angiogenesis markers upon EVs treatment. To study EVs as drug carriers, a more complex multicellular cultures and organoids from cancer stem cell populations have been developed. Such strategies provide a closer response to in vivo physiology observed responses. They are also the best models to understand the complex interactions between different populations of cells and the extracellular matrix, in which tumor-derived EVs modify epithelial or mesenchymal cells to become protumor agents. Finally, the growth of cells in 3D bioreactor-like systems is appointed as the best approach to industrial EVs production, a necessary step toward clinical translation of EVs-based therapy. ; The review is supported by Spanish Ministry of Science and Innovation, within the national Plan RTI2018-094969-B-I00, and Excellence Severo Ochoa grant Innovative Research Grant (SEV-2016- 0644), and by the European Union's Horizon 2020 research and innovation program, grant number 860303.

Early View: Online Version of Record before inclusion in an issue. ; Carotenoids are lipophilic plastidial isoprenoids highly valued as nutrients and natural pigments. A correct balance of chlorophylls and carotenoids is required for photosynthesis and therefore highly regulated, making carotenoid enrichment of green tissues challenging. Here we show that leaf carotenoid levels can be boosted through engineering their biosynthesis outside the chloroplast. Transient expression experiments in Nicotiana benthamiana leaves indicated that high extraplastidial production of carotenoids requires an enhanced supply of their isoprenoid precursors in the cytosol, which was achieved using a deregulated form of the main rate‐determining enzyme of the mevalonic acid (MVA) pathway. Constructs encoding bacterial enzymes were used to convert these MVA‐derived precursors into carotenoid biosynthetic intermediates that do not normally accumulate in leaves, such as phytoene and lycopene. Cytosolic versions of these enzymes produced extraplastidial carotenoids at levels similar to those of total endogenous (i.e. chloroplast) carotenoids. Strategies to enhance the development of endomembrane structures and lipid bodies as potential extraplastidial carotenoid storage systems were not successful to further increase carotenoid contents. Phytoene was found to be more bioaccessible when accumulated outside plastids, whereas lycopene formed cytosolic crystalloids very similar to those found in the chromoplasts of ripe tomatoes. This extraplastidial production of phytoene and lycopene led to an increased antioxidant capacity of leaves. Finally, we demonstrate that our system can be adapted for the biofortification of leafy vegetables such as lettuce. ; This work was funded by the European Regional Development Fund (FEDER) and the Spanish Agencia Estatal de Investigación (grants BIO2017‐84041‐P and, BIO2017‐90877‐REDT), Generalitat de Catalunya (2017SGR‐710), and European Union's Horizon 2020 (EU‐H2020) COST Action CA15136 (EuroCaroten) to MRC. We also acknowledge the financial support of the Severo Ochoa Programme for Centres of Excellence in R&D 2016‐2019 (SEV‐2015‐0533) and the Generalitat de Catalunya CERCA Programme to CRAG. This work also benefited from the Biophysics Platform of I2BC, supported by iBiSA and by the French Infrastructure for Integrated Structural Biology (FRISBI) ANR‐10‐INBS‐05. TBA was funded by a Carlsberg Foundation fellowship. BL is supported by grants from the CSIRO Synthetic Biology Future Science Platform and Macquarie University. LM is supported by La Caixa Foundation PhD INPhINIT (ID 100010434) fellowship LCF/BQ/IN18/11660004, which received funding from the EU‐H2020 (MSCA grant 713673). STM is supported by a PhD fellowship from the Spanish Ministry of Education, Culture and Sports (FPU16/04054). ; Peer reviewed