Peer review of: ITGB3-mediated uptake of small extracellular vesicles facilitates intercellular communication (2021)


 
 

Reviewer #1 (Remarks to the Author): 
 
Although I feel that this 2nd revision is an incremental improvement on the previous revision, 
there are still a number of questions that remain unanswered – such as the mechanism by which 
HSPGs affect EV uptake, or the identity of cellular compartments in which EVs accumulate in 
recipient cells. Furthermore, I feel that it is hard to draw any meaningful conclusions from some of 
the confocal microscopy data presented in the paper. For these reasons, I am still cautious to 
recommend this manuscript for publication in Nature Comms. as it currently stands. 
 
Specific points: 
- The title needs to be modified, as the paper does not address the effect of ITGB3-mediated EV 
internalisation on breast cancer metastasis. Even though previous work from the authors 
suggested that ITGB3 is required for breast cancer metastasis in the mouse lung, the title should 
only refer to work in the present manuscript. Instead of “breast cancer metastasis”, consider using 
“breast cancer clonal growth” or a similar term. 
- Throughout the text, there seems to be confusion regarding the term “recycling of integrins”. In 
the trafficking field, “receptor recycling” refers to the “return” of receptors from intracellular 
compartments (e.g. recycling endosomes) to the plasma membrane. In many parts of the text 
(e.g. abstract line 38, introduction line 95, results line 204 and 307, discussion line 379), the 
authors use the term when instead should be referring to endocytosis/internalisation of integrins. 
This should be changed accordingly. 
- The authors need to plot quantifications of EVs with sizes ranging 150 – 200 nm. Although the 
authors claim in the text that ITGB3-kd leads to a reduction of EVs within this range in the 
condition media, this is not shown in Fig 4C. 
- The quantification of colocalisation between labelled EVs and recipient cell EEA1 (Supp. Fig. 9) 
revealed that the loss of ITGB3 in recipient cells does not influence the accumulation of EVs in 
early endosomes. Is it fair to say these observations do not follow the authors conclusions nor the 
model presented in Fig. 6H, where ITGB3 is suggested to mediate dynamin-dependent 
internalisation of EVs? Would one not expect the levels of EVs in early endosomes to be affected 
by ITGB3-loss? The observation that ITGB3-kd does not affect accumulation of EV in early 
endosomes needs to be added to the results text, and we advise the authors to discuss this in the 
discussion. 
- Invert lookup table in the confocal microscopy images, so signal is displayed in black and 
background in white. 
- Add in supplemental the FACS plots (I think that these had been in a previous version of the 
paper) from which the bar charts for EV internalisation come from. Also display how the gating 
was setup. 
- Add citation in line 107 and line 300. 
- Labels in Fig. 3C are wrong? 
- For the story to flow in a more logical manner, we recommend that the authors move the data 
regarding the analysis of ITGB3-dependent EVs (Figure 4 and 5) to the end, following the 
requirement of FAK for EV internalisation, and change the text accordingly.