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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.