Exosomes are extracellular vesicles that are thought to mediate cell-to-cell communication in eukaryotes. Briefly, exosomes are 50-100 nanometer (nm) sized vesicles produced by the endosomal system. They are exported out of the cell and can be found in every bodily fluid: plasma, saliva, milk, urine and more. These vesicles then enter recipient cells, and the cargo they carry (proteins, RNA molecules and lipids) modulate the physiology and/or gene expression of the recipient cell. Exosomes catch a lot of attention lately because of their clinical significance. First, exosomes might be used as biomarkers for some diseases (most importantly tumors). Second, they are being considered for therapeutics as a delivery system.
Although the formation and secretion of exosomes is fairly understood (though we are just beginning to understand how some cargo is being packed), it is less clear how exosomes enter recipient cells. Even less clear is what happens to them once they enter the cell.
I myself have been concerned with the math of exosome-mediated communication. In most papers, exosomes are isolated from the donor cells (e.g. from culture media), then concentrated before applying them on top of recipient cells in vitro. However, it is not always mentioned what is the ratio of exosomes/cells and it is also unclear how many exosomes actually enter the cells. Furthermore, it is still unclear how much cargo is carried by a single exosome.
There have been a few papers which looked at how exosomes are internalized, and where they go. Some papers suggest exosomes are endocytosed in a receptor-mediated process, others suggest macropinocytosis or phagocytosis whereas others suggest fusion with the plasma membrane.
The subcellular fate of the exosomes is still unclear. I know of two papers which suggest that they end up in lysosomes (cited below) but how do they get there and what happens to the cargo is still unclear.
Speaking of cargo, to my knowledge there have been only two papers which tried to estimate the number of miRNA molecules per exosome(cited below). One paper suggests a single molecule (of a specific miRNA) per 100 to 10,000 exosomes (i.e. only one out of 100-10,000 exosomes contain one molecule f that specific miRNA). The wide range is due to differences in miRNA species and source of the exosomes. A second paper that only looked at a single miRNA species in a particular cell type was more optimistic, estimating 10-60 molecules per exosome. Still, with such low numbers, how many exosomes need to enter recipient cells and release their cargo to affect gene expression?
Well, I don’t have answers to all these questions, but a new paper published recently tries to shed some light on the journey of the exosomes. Here, the authors labeled exosomes by expressing CD63 fused to GFP in the donor cells. CD63 is a common “marker” for exosome. Although recently it was shown that there may be a large variety of extracellular vesicles with different properties (size, density), including different proteomes (cited below), CD63 still seems like a good marker for “true” exosomes.
They isolated the exosomes by a series of centrifugations/ultra-centrifugations and filtrations and the exosomes were quantified by FCS. Now that they have pure exosomes, they applied them onto the recipient cells and started to quantify their uptake by imaging. They first noticed that exosomes start entering the cells within minutes after addition, with 80% entering the cells by 2 hours. Uptake was time and dose dependent, but it always reached saturation. This indicates that there is a balance between uptake and degradation of exosomes or that after a while new exosomes cannot enter the cell.
Now let us look at the numbers. The number of internalized exosomes per cell was dependent on exosomes concentration, duration, and number of recipient cells. The average numbers ranged from <10 exosomes/cell to ~30 exosomes/cell. If we look at single cells, at very low percentage (<0.1% of cells) the number of exosomes/cell exceeded 100. The authors chose to quantify the exosomes in picoMolar units. In figure S1a they used 10pM of exosomes. If my math is correct, then this translates to 6×1012 exosomes/liter. Assuming 0.1ml volume in their 96-well experiment, this means 6×108 exosomes applied to a few thousand cells. So, for 6000 cells we get a ratio of 100,000 exosomes per cell. With an average of 10 internalized exosomes/cell this gives a yield of 0.01% for exosome internalization.
So here is something to think about – what is the biological significance for a process which is only 0.01% efficient? Is it due to the assay? Are exosomes being damaged somehow during their isolation/application onto the cells? If each exosome carries only a few microRNA molecules (does each exosome carry only one species? Or does each exosome carry multiple species, a few copies each? – it is unclear) what affect can 10 exosomes have on the cell physiology?
The major emphasis of this paper is the mechanism of entry and what happens next. Amazingly, it looks as if exosomes attach to filopodia and either surf on them towards the cell (25%) or they are pulled or grabbed by filopodia (3%, 1%, though this is an underestimation due to limitations of imaging). Look at the images. It looks awesome!
(Note that all the movies and tracking analysis was done with application of 100pM of exosomes – much higher than the concentrations used to quantify entry in Fig 1 a-c & S1 a-b. It is not clear to me why they used such a high concentration when 30pM , even 15pM, was already saturating. )
The most important finding here is that they see, just like in previous publications, that exosomes end up in lysosomes. However, here, they tracked exosomes inside the cell more carefully. First, they show that the majority of exosomes are actually found inside endosomes (fluorescent and EM images show that). Then they show that exosomes have brief interactions with the ER (seconds to a few minutes). Since ER is the region where most mRNAs are being translated, it is reasonable to assume that the RNA cargo will go there. However – how does the cargo pass through 4 membranes (exosome is a double-membrane vesicle and endosome is a double membrane vesicle)? The authors do not really discuss that.
Last, after 48hr, 50-60% of exosomes ended up colocalizing with lysosomes, where, presumably, they are being degraded.
I found this paper very interesting, and I was happy to see that the data is quantitative, rather than qualitative. But the results here raised more questions about the effects exosomes may have on recipient cell physiology and the biological consequences of this form of cell-to-cell communication.
Next month we have a meeting here at the Weizmann Institute Extracellular Vesicles:
friends and foes. The deadline for abstract submission is over, but you can still register to attend. [Update: I’m going to present a poster +3min talk at this meeting]
[Update: see also the post on counting single-cell exosome secretion ]
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