Studying the Fate of Tumor Extracellular Vesicles at High Spatiotemporal Resolution Using the Zebrafish Embryo
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Abstract
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This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/7041767.
The paper presents Zebrafish as a new and appropriate model system to explore the dynamics of tumor extracellular vesicles (EVs) from their source of biogenesis to their sink. It beautifully links the pre-established advantages of Zebrafish model system to the lacunae in the field of circulating EV visualization. While it is satisfying to see the authors establish similarities between Zebrafish and human melanoma EVs in terms of their protein content and geometries, it would have been prudent to perform a mass spectrometric analysis to assess the lipid content of the EVs as well (Fig. 1). Due to the differential lipid compositions of zebrafish and human plasma membranes, their respective EV …
This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/7041767.
The paper presents Zebrafish as a new and appropriate model system to explore the dynamics of tumor extracellular vesicles (EVs) from their source of biogenesis to their sink. It beautifully links the pre-established advantages of Zebrafish model system to the lacunae in the field of circulating EV visualization. While it is satisfying to see the authors establish similarities between Zebrafish and human melanoma EVs in terms of their protein content and geometries, it would have been prudent to perform a mass spectrometric analysis to assess the lipid content of the EVs as well (Fig. 1). Due to the differential lipid compositions of zebrafish and human plasma membranes, their respective EV membranes might significantly vary in their lipids, since EVs can arise from plasma membrane as well as endosomal compartments. Lipid compositions might also affect the fusion ability of the EVs at their sink, which as explained in the paper, is crucial for their tumorigenicity. In addition to the comparative protein analysis already performed, lipid-based analysis would further increase the translatability of this system.
Standardizing the use of MemBright labeling as a way to mark EVs was inspired. While this approach is easy, economical, and adaptable, one tends to question its specificity. Though the authors have attempted to provide solid parameters for establishing labeling specificity, it would indeed be meritorious to use proteins specific to particular EVs to confirm one's results, especially while testing distant traveling EVs (Fig. 7).
Experiments conducted towards analyzing dynamics of EVs in the blood flow require 100 nm beads to be individually or co-injected as a control. It is necessary to test if the flowing, rolling, or arrest dynamics seen with EVs at different points in the vessel were characteristic of EVs or if such dynamics are coincidentally seen with objects of similar shape and sizes. Claims about the adhesive properties of EVs cannot be substantiated without this control experiment (Fig. 3). Experiments conducted in figure 4 to understand EV uptake by various cell types also require bead controls as bead phagocytosis in macrophages and endothelial cells is well-established and could indicate coincidental results seen with EVs. Due to these reasons, claims made in figures 3, 4, and 5 fall short of being conclusive.
Confusion between the mode of EV endocytosis being filopodia surfing or macropinocytosis could be resolved by using specific inhibitors of macropinocytosis such as imipramine (Fig. 6). This could be repeated in the cell culture-based experiments as well to establish macropinocytosis as the mode of EV uptake.
Lastly, it might be beneficial to delve deeper into the consequences of EV internalization by macrophages and its contribution to metastasis. What happens to the biochemical and biophysical makeup of macrophages upon continuous EV internalization and what is the limit on their ability to clean up these particles? Using pH-sensitive fluorophores like AcidiFluor or pHluorin to specifically label proteins and EVs of interest might also provide insights into their uptake and further transport through the endosomal system towards acidic lysosomes.
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This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/3662409.
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 …
This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/3662409.
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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