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

Reviewers' comments: 
 
Reviewer #1 (Remarks to the Author): 
 
This manuscript from the yCajal lab describes experiments aimed at determining a role for avb3 
integrin in internalisation of extracellular vesicles (EVs) . The authors have found that knocking 
down b3 integrin reduced the level of EV uptake by MDA-MB-231 cells. They show that activation 
of focal adhesion kinase (FAK) and dynamin are also required for EV uptake. Inhibition of this EV 
uptake process seems to reduce the clonal growth of MDA-MB-213 cells, possibly because this 
renders the cells less sensitive to growth promoting factors in the secretome. 
The authors have identified an interesting phenomenon, and the findings would be of interest to 
the readership of Nat. Comms. However, as the paper stands, the data do not support the 
conclusions drawn by the authors. Moreover, the paper needs substantially more mechanistic 
insight into b3 integrin –dependent EV internalisation before acceptance in Nat. Comms. is 
justified. 
 
Major points: 
1. The FACS-based assay to measure uptake of fluorescently labelled EVs does not discriminate 
between EVs that are attached to the cell surface and those that are internalised into the cell. 
High-resolution confocal microscopy to directly visualise labelled EVs inside cells/endosomes would 
go a long way to support their findings. The authors should also visualise EV internalisation in cells 
where dynamin and ITGB3 have been inhibited/knocked down to determine whether these factors 
are indeed required for EV internalisation and not simply binding of EVs to the plasma membrane. 
2. To ensure that the defects in EV internalisation observed in Fig 2 are not simply due to reduced 
levels of ITGB3 at the cell surface, the surface expression of ITGB3 following treatment with 
FAK14, Dyngo, PitSTOP and Heparin should be measured. Moreover, as FAK14 treatment occurs 
over a long time-period, the total levels of b3 integrin should also be measured for this treatment. 
3. The small molecule inhibitor of dynamin Dyngo4 has reported off-target activities that inhibit 
fluid-phase endocytosis. There is a danger that the results in Fig2B and Fig5A linking EV 
internalisation/FAK activation to dynamin, are artefactual. Therefore, EV internalisation (Fig2B) 
and EV-induced FAK phosphorylation (Fig5a) should be repeated with knockdown of one or more 
dynamin isoforms and/or overexpression of dominant negative dynamin (Dynamin-2K44A). 
Similarly, experiments using the FAK inhibitor FAK14 should be repeated with FAK knockdown 
where possible. 
4. Generally, there is a lack of data providing mechanistic detail on the role of HSPGs and b3 
integrin during EV internalisation. This study would benefit greatly from a set of experiments using 
high resolution microscopy to dissect: a) The co-localisation of labelled EVs with b3 at the cell 
surface. And the requirement of HSPGs for this; b) The internalisation of EV-b3 complexes/foci 
within endosomes, for instance EEA1-posotive endosomes; c) The recruitment of FAK to EV-b3 
positive foci, and the phosphorylation-status of FAK within these structure. 
5. The authors show that treatment with the FAK inhibitor FAK14 reduces EV internalisation 
(Fig2C) and conclude that EV uptake requires FAK activity. siRNA-mediated knockdown of FAK 
should achieve similar results. Moreover, it would be predicted that FAK activation/constitutively-
active FAK would rescue EV uptake by shITGB3 cells, is this the case? 
6. From the NTA profiles shown in Fig3A it appears that shCONTROL cells release far more EVs 
than shITGB3 cells, however, when normalised to cell number (Fig3C) the opposite is the case. 
This suggests that there are far fewer shITGB3 cells in the EV preps and the authors should 
provide an explanation for this. Do shITGB3 cells grow more slowly than shCONTROL cells? As 
mentioned above, this should this should be formally tested as the results of this are crucial for the 
interpretation of additional experiments, e.g. the colony formation assays in (Fig1) and the IMR90 
co-culture in (Supp Fig 4). 
7. Throughout this study, western blots are performed on EV lysates without the relevant whole 
cell lysate samples. This means that all data concerning the constituents of shCONTROL vs 
shITGB3 EVs are difficult to interpret. Molecular weight markers are absent from all western blot 
images and should be included. 

8. The authors show compelling evidence that ITGB3 promotes CD81/TSG101-containing exosome 
biogenesis. However, the paper would benefit from further characterisation of the EV populations 
released by shCONTROL and shITGB3 cells. For instance, sucrose or Iodixanol gradients should be 
used to separate vesicle populations prior to western blotting. 
9. The claim that EV-induced activation of FAK is a driver of exosome biogenesis needs to be 
further supported. The mechanism for this is unclear. The authors should address this by 
investigating the role of ITGB3/FAK in regulating; a) the biogenesis of ILVs in the MVB; b) MVB 
dynamics (localisation, trafficking and exocytosis) 
10. The authors show that dynamin activity is required for FAK activation following EV treatment. 
Are additional components of integrin signalling (Src, Akt, Erk) also activated following EV 
treatment? Moreover, does FAK signalling play a role in colony formation (Fig1A). For instance, do 
conditioned medium-treated cells have higher levels of phospho-FAK? Do FAK inhibitors impair 
colony formation in the presence of conditioned media? 
11. In Fig 5B the authors use TCA precipitation of cell culture supernatant to show that the levels 
of secreted CD81 are reduced following FAK inhibition. From this they conclude that FAK activity is 
responsible for driving exosome biogenesis and secretion, but these data are somewhat 
preliminary. It is unclear, for instance, why they have not performed western blots on EVs isolated 
from FAK14-treated cells. This should also be supported with NTA of EVs isolated in the presence 
of the FAK14 to determine. If the authors are right, this should lead to similar changes in EV size 
distribution and concentration as those observed in the shITGB3 cells. 
12. Generally, the conclusions outlined in the discussion text and in Fig5C are overstated with only 
preliminary supporting evidence. For instance, the conclusion that FAK activation following EV 
internalisation promotes syntenin-ALIX-ESCRT recruitment on MVBs is totally unsupported. Either 
the evidence should be provided, or the text should be toned down. 
 
 
Minor points 
1. The authors show compelling evidence that the EV contents of conditioned medium contribute 
towards colony formation of MDA-MB-231 cells, and this is subsequently attributed to their uptake 
of extracellular vesicles, likely exosomes. It is unclear whether large microvesicles/ectosomes or 
small EVs within the conditioned media are responsible for this however. This could be addressed 
by assessing the colony forming capacity of CM from which only microvesicles/ectosomes have 
been depleted, for instance, by subjecting the conditioned media to a 10,000 x g spin. 
2. In Fig 2A the authors maintain target cells at 4 oC during incubation with EVs. The aim of this 
experiment is to assess whether EV internalisation is an energy dependent or a passive process. 
The design of this experiment is problematic as passive fusion of membrane compartments is likely 
to be severely inhibited at 4 oC due to reduced membrane fluidity. Can the authors show that 
passive fusion of membranes is uninhibited at 4 oC? 
3. Fibronectin is a described EV cargo and fibronectin-containing EVs have been shown to support 
formation of cell adhesions (Sung et al, Nat. Comms. 2015). It is a possibility that the authors 
may be observing ECM-induced FAK activation following EV treatment. Do the EVs isolated form 
their cells contain fibronectin or other ECM components? If this is the case, the involvement of 
these ECM components in their EV-induced FAK activation should be investigated. 
4. Line number 159 describes Dyngo-4a as a “small molecule inhibitor that blocks GTPase 
activity”. Reference 46 is incorrect- this reference from 2009 is for the drug Dynole where as 
Dyngo-4a was developed in 2013. Dyngo-4a reduces GTPase activity of dynamin and is believed to 
act by reducing its ability to oligomerise. The reference they require is Traffic. 2013 
Dec;14(12):1272-89. ‘Building a better dynasore: the dyngo compounds potently inhibit dynamin 
and endocytosis’ McCluskey A et al. 
 
 
 

 

Reviewer #2 (Remarks to the Author): 
 
In this study, Fuentes et al showed that ITGB3 knock-down inhibits extracellular vesicle- uptake ( 
through dynamin-dependent endocytosis), change the composition and size of vesicles present in 
the culture media of ITGB3 KD cells, and that ITGB3+ vesicles are required for colony formation of 
breast cancer cell. 
 
The authors propose that ITGB3 controls both exosome uptake and release through activation of 
the Focal Adhesion Kinase, and speculate that endosome could be the signalling platform. 
 
The experiments are well designed and executed, and well-presented. The manuscript is clear and 
well written. hoverer, the statements are very strong and not always supported by the data. 
 
I have two major concerns that needs to be addressed (especially the first one): 
1) 
In Figure 2a and B, the authors use a 4 degrees C temperature block to inhibits energy dependent 
internalization. To my knowledge, ligand-receptor interaction is not abolished at 40C. a good 
example is the binding of LDL to its receptor that still occurs at 4 degrees C, although LDL 
internalization is abolished (Brown and Golstein PNAS 1974). Therefore, one would expect to 
detect fluorescently labelled exosomes at the surface of the acceptor cells at 4 degrees C. This is 
not the case in fig2 a and b. This suggests to me that ITGB3 does not contribute to exosome 
docking at the surface of the cell, which contradicts author's statement and conclusion. ITGB3 may 
be involved somehow in EV endocytosis, but does not seem to be sufficient to enable docking of 
exosomes to the target cell. This diminishes greatly the finding of the paper. 
2) 
The authors concluded that ITGB3 controls also exosome release because they found that 
exosomes emanating from ITGB3KD cell are different in size and composition. Another 
interpretation is that ITGB3 is dedicated to the internalization of a specific subtype of exosomes. 
This population would corresponds to small EVs (50 -100nm) that would be depleted of escrt 
complex and other hits found in the paper. This would also explain why in fig2A and B vesivle-
internalization is reduced by a factor r 2 (i.e the larger EV are still internalized). The authors might 
consider discussing this possibility. 
 
To conclude, the first point is an important caveat that needs to be addressed by the authors, 
because it change dramatically the conclusion made by the authors.