Hi, my name is Kate Timms and I am a Scientific Content Writer at Izon Science
and today I’m going to be talking to you about types of extracellular vesicles.
Back when I started my PhD on extracellular vesicles in 2014,
it was common to refer to all small extracellular vesicles as exosomes.
We simply didn’t really know better as a field.
Even when I submitted my thesis, the markers I used
and the morphology I showed using electron microscopy were thought
to be characteristic of exosomes rather than EVs in general.
At that point though, the field was starting to get
significantly more cautious about the use of the term ‘exosome’
and I was quizzed about the identity of my EVs in my viva.
Fast forward to 2022 when my PhD research was finally published.
I wouldn’t have dreamed of calling the extracellular vesicles I had studied
exosomes, despite referring to them as that at the time.
And I had mass spectrometry showing that at least
some of the vesicles were enriched for endosomal proteins.
So why the change?
Why did I almost cringe at saying the word ‘exosome’?
Well, change is essential in science.
The very foundation that modern science is built upon
dictates that we must be malleable to changing our hypotheses
and what we think we know about the world based upon newly revealed knowledge.
The field of extracellular vesicles is – or at least is generally trying to – do just that.
As with every burgeoning field, things seemed complex but relatively simple at first.
EVs were divided into three categories back then.
The smallest were exosomes,
the middle sized were microvesicles and the largest were apoptotic bodies.
That’s the diagram I drew for my PhD thesis.
But it simply isn’t the full story.
As we dug deeper into these fascinating vesicles
we found more and more variation, more subtypes, and less certainty.
Now we know that the number of EV subtypes is up there in the dozens,
perhaps even higher.
This schematic shows a more complex version
of affairs than I reported in my PhD thesis.
The old favourites are still there.
You can see exosomes forming as intraluminal vesicles in late
endosomes, turning them into multivesicular bodies
which can then fuse with the plasma membrane to release their cargo,
now called exosomes.
Microvesicles are present too, budding from the plasma membrane,
though now we show their range of sizes as overlapping with that of exosomes
as well as larger vesicles.
Cancer cell microvesicles
are now known as oncosomes and are perhaps even larger.
Apoptotic bodies are still there too, though there is more variation
in apoptotic-cell-derived EVs than we previously thought.
There are also the migrasomes, left behind by migrating cells,
and the secretory autophagosomes which may contain whole organelles.
Finally there are cell-type specific EVs such as syncytial knots,
which are nuclei-filled vesicles
which pinch off from the placental syncytiotrophoblast.
And those are just the ones we could fit onto this diagram.
There are likely many more EV subtypes yet to be discovered.
With so many types of EVs,
you may think that there would be a plethora of markers to tell them apart.
Unfortunately, that isn’t the case.
Evidence from the literature is conclusive:
truly specific EV markers likely don’t exist.
And this isn’t without effort from the field.
A marker for exosomes is somewhat of a Holy Grail,
yet every promising candidate so far has eventually been discounted.
The endosomal sorting complexes required for transport
(or ESCRT proteins) were long thought to be specific to exosomes,
especially given that their name implied that they were endosomal.
However, it turns out that ESCRT complexes I, II and III are all involved
in the budding of microvesicles at the surface, discounting them –
including the long-hailed exosome marker TSG101 –
from being exosome specific.
The same is true for syntenin-1 and Alix,
both of which are present in EVs of different subtypes.
However, the ESCRT-0 complex may be endosome
specific, making its components potential markers of exosomes.
Indeed, knocking down two of the ESCRT-0 proteins
reduced the release of CD63+ve small EVs.
And CD63 is exosomal, right?
Wrong.
Unfortunately.
At least according to a recent study.
Tetraspanins like CD9, CD81 and CD63 were long touted as exosome markers,
but they have also been found to also be present in other EV subtypes.
CD63 positive EVs which also carry CD9 are likely
membrane budding and are therefore microvesicles.
However, it has also recently been reported
that EVs which are CD63 positive but CD9 negative may indeed be exosomes.
But how can you be sure that is true for every EV matching those criteria?
Couldn’t a membrane-budding vesicle positive for CD63
just by chance miss out on grabbing its own CD9 molecule at the plasma membrane
and so unwittingly masquerade as an exosome?
Given the ambiguity here,
CD63 is not a very attractive exosome marker.
Using a negative marker in CD9 is not attractive either.
After all, absence of evidence is not evidence of absence.
However, all may not be lost.
LAMP proteins may hold the answer.
LAMP proteins are used as markers of the lysosome and certain endosomes,
making them an attractive candidate for an exosome marker.
Especially, as they were found to be substantially enriched in CD63
positive EVs as compared to CD9 positive EVs.
Even so, we would be cautious to call anything a true exosome marker.
Even our current understanding tells us that EVs are more complex than that,
and there are probably no hard and fast rules.
Especially between different cell types.
At least for now, an exosome’s biogenesis needs to be seen to be believed.
But what about tools to study exosomes?
GW4869 is an inhibitor of the neutral sphingomyelinase,
which is thought to be required
for the ceramide-dependent budding of intraluminal vesicles.
This has led it to being billed as an exosome inhibitor.
However, a recent study showed that GW4869 has little to no effect
on the secretion of CD63+ve and CD9-ve EVs, which are likely exosomes.
This led to the authors of this study to warn caution
when using GW4869.
What does all this uncertainty mean for the EV field?
Well for starters, it suggests that using the umbrella term
‘extracellular vesicles’ is likely the safest
and most accurate way to describe your vesicles.
Exosome may be a great word that the people outside of the field recognise,
but using it in your manuscript title or abstract doesn’t
actually get you any more citations.
Despite the prevailing wisdom that the term garners more attention.
Secondly, it means that we should all be a bit
more cautious when choosing markers and tools to study our vesicles.
More research is needed to further characterise EV subsets.
Tunable Resistive Pulse Sensing,
such as the Exoid, is a great place to start.
With the Exoid,
you can measure each vesicle individually in your heterogeneous EV sample,
allowing you to visualise subtypes of EVs by their size and charge.
Here at Izon Science, we can help you in starting your journey
to understand the true complexity of your EV samples,
from rapid and efficient isolation through to characterisation.