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



Reviewers' comments: 
 
Reviewer #1 (Remarks to the Author): 
 
This revision has significantly improved the paper and I appreciate the authors taking my 
suggestions on board. I feel, however, that some issues remain as the paper stands, particularly 
regarding the visualisation of internalised EVs in recipient cells by confocal microscopy. 
 
Main points: 
Imaging – 
1. 
a) In order to overcome difficulties in distinguishing between donor and recipient ITGB3-positive 
structures using confocal microscopy (Supporting Figure 1), the authors could repeat these 
imaging experiments using EVs derived from ITGB3-knockdown cells. This could work as loss of 
ITGB3 in donor cells did not affect recipient cell-EV uptake (Figure 1). The experiment displayed in 
rebuttal letter Supporting Figure 3 could also be repeated using EVs derived from ITGB3-
knockdown cells. 
 
b) In supporting Figure 4, the authors were not able to demonstrate the colocalisation between 
labelled EVs and p-FAK in recipient cells. Although we agree with the authors that confocal 
microscopy is at the limit of resolution for detection of single EVs, it is well established that EVs 
accumulate within the endosomal system upon internalisation in recipient cells, and EV-positive 
vesicular structures can be usually clearly visualised using such microscopy techniques. Are the 
PKH26-positive structures associated with focal adhesions? Live cell imaging of recipient cells 
transiently expressing GFP-FAK +/- ITGB3si in the presence of labelled EVs would help 
characterising the temporal events of FAK activation downstream of EV uptake, and to answer 
whether EVs colocalise with FAK in recipient cells. 
 
c) In the rebuttal letter the authors include images of labelled EVs with ITGB3 and these images 
look a lot better than the ones shown in figure 3d. I think the other points made in the rebuttal 
letter should also be included in the paper. Also fig 3d would benefit from showing each channel 
separately black on white background as this would be much clearer. 
 
 
2. The authors show that depletion of EVs from CM impairs its ability to promote clonal growth in 
MDA-MB-231 cells (Fig. 1). However, instead of experimentally testing whether the incubation of 
recipient MDA-MB-231 cells with EVs stimulate clonal growth, the authors evaluated the migration 
of recipient IMR90 fibroblasts (Supp. Fig 9). Although these data are interesting, it is not clear why 
this EV-function readout (i.e. cell migration) is used at this stage. Does the incubation of MDA-MB-
231 cells with EVs stimulate their clonal growth? Is ITGB3 in recipient cells required for this? 
 
3. The link between syndecans and the mechanism of EV uptake that the authors described is still 
very weak. Perhaps the MS data for the proteome of EVs might inform on whether a particular 
syndecan is highly abundant in MDA-MB-231-EV preps, and if so, it would be of interest to 
evaluate the effect that knocking-down such syndecan in donor cells has on EV uptake. 
 
4. The TITLE is misleading, as the authors did not study the integrin recycling throughout the MS. 
Instead, the phenotype regulating EV uptake is coordinated with integrin internalisation, and the 
title should reflect this. 
 
Other points: 
1. The order of presentation of the data and the organisation of the paper renders it difficult to 
read and understand. For example, line 181 pertains to figure 3c-d then the next figures that are 
mentioned are in the order of Fig2b, sup fig 8a, fig2e, fig 2f then figure 4a. Also Fig. 3b is not 
mentioned in text. This is similar in the supplementary data which should be in the same order in 

the text and linked to the same data figure. 
2. The choice to use IMR90 cell exosomes in figure 1 is not explicitly explained. The text needs a 
sentence or two to explain the reason for using these cells in relation to cancer. 
3. Supplementary figure 6 needs to be labelled as such and in a separate data file 
4. Supplementary figure 5A needs labelling control and shITGB3 
5. The authors should discuss work from Meisner-Kober lab showing that filopodia mediate EV 
uptake (https://rupress.org/jcb/article-lookup/doi/10.1083/jcb.201506084) 
 
 
 
 
 
Reviewer #2 (Remarks to the Author): 
 
In the revised manuscript, the authors invested a large effort to further characterize the EV 
behaviour/fate at 4 degrees C (new fig2a and 3). The data and the study has been greatly 
improved. They also satisfyingly answers comment raised in point 2 of the initial review. 
Concerning the initial point 1 (interpretation of lack of surface binding at 4 C). This reviewer 
agrees that itg3 participates in EV uptake and is convinced by the data (especially new fig2a and 
3). The explanations provided in the response letter are also clear. There is no strong EV binding 
at the cell surface at 4C. Again, this suggests (to this reviewer) the lack of BONA-FIDE EV-receptor 
because protein-protein interaction are normally maintained at 4 C. This reviewer feels that it 
should be clearly stated in the discussion that ITG3 participates in EV uptake but that the lack of 
surface binding at 4C suggests that ITG3 is not a bona fide EV receptor. This could be emphasised 
through comparison with the LDL receptor, using the same language used by the authors in the 
response letter. 
To summarise, data are convincing, the model in fig 6 still stands especially the dynamin 
dependency, but the lack of strong statement discussing the point raised above is a high risk to 
mislead readers and the scientific community who may conclude that ITG3 is indeed a specific 
bona fide EV receptor.