1. Reviewed by PREreview
    Dear authors,please find enclosed our review report on your preprint asdiscussed during our preprint JC session at the IGDR. We hope that you will find it useful.
    The authors present a novel in vivo technique to track extracellular vesicles (EVs) using zebrafish embryo. They used this technique to track tumor EVs dynamics and fate in a living organism. With a nicely putted introduction, they then proved with different experiments that MemBright has brighter signal compared to other conventional techniques and reduce staining artifacts.
    Main concerns :When analysing the behaviour of EVs injected in the bloodflow, we would have liked to see beads co-injected as a control. This would enable to determine whether the trajectory of the EVs and the cells in which they are uptaken are in fact specific to the EVs and not to any 100nm object present in the blood. Without this control, conclusions of figures 3, 4 and 5 cannot be fully trusted.Improvements are needed to test the possibility to use zebrafish to track tumors Evs. First of all, testing a range of Evs concentration could be great in order to avoid the possibility that macrophages uptake is due to huge amount of Evs compared to physiological case. We propose to test this hypothesis by correlating number of macrophages events uptake upon changing in Evs concentration. Secondly, we wish to go deeper in the understanding of macrophages roles in EVs uptake. Could the authors demonstrate that EVs are targeted to lysosomes in order to be cleared? A better characterization of EVs uptake into lysosomes is also required, using CLEM for example. To conclude, should be great to show that "local" EVs are targeted to lysosomes like the external ones.
    Minor concerns: The article could benefits from a reduce number of figures in each panel. We advise the authors to select the most informative figures that clealy illustrates one key message. We also think that the titile could be improved to clearly highlight the emergence of this novel technique, example : "MemBright: a new technology to study EVs...".

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