NEW YORK (GenomeWeb) – A new report from a team led by Fred Hutchinson Cancer Research Center (FHCRC) scientists indicates that exosomes, on average, harbor significantly fewer copies of microRNAs than previously believed, adding a new wrinkle to ongoing research into the role of these cell-derived vesicles as possible vehicles for intracellular miRNA transfer.
Importantly, the findings only reflect the absolute abundance of miRNAs within a population of exosomes, leaving open the possibility of exosome subpopulations that may carry large numbers of miRNAs, said Muneesh Tewari, a former FHCRC researcher who led the study who is currently an associate professor at the University of Michigan.
"We still don't have a technology with which we can purify one exosome and do a single-exosome microRNA quantification," he told Gene Silencing News this week. But with such tools under active development, the next step in this field will be to look for those subsets of exosomes and determine if they are biologically relevant.
For a number of years, reports have been appearing in the literature that point to the presence of miRNAs in exosomes, leading some to speculate that they represent a system for cell-to-cell communication.
And because exosomes often feature cell surface proteins derived from their cell of origin, which can be recognized by cell surface receptors, internalized by recipient cells, and result in transfer of the exosome contents, exosome-mediated miRNA transfer has also been proposed as a possible intracellular regulatory mechanism.
Meanwhile, some groups have been looking into whether miRNA-bearing exosomes could serve as biomarkers given the association between miRNAs and diseases such as cancer.
Yet current mechanistic models of this process lack detail, and "the physiologic significance of this paradigm is not yet established," the FHCRC team wrote in its study, which appeared in Proceedings of the National Academy of Sciences.
"Quantitative evaluation of key components is fundamental to testing the validity of any model, and knowledge of the stoichiometry of miRNAs and exosomes — i.e., how many molecules of a given miRNA are carried by an exosome — would provide insight into the requirements and limits of miRNA-based intercellular communication," they added.
To that end, Tewari and his colleagues set out to determine the degree to which extracellular biomarker miRNAs are associated with exosomes, focusing on ones prepared from plasma of prostate cancer patients in light of several studies that found miRNAs within exosomes excreted from cancer cells.
In their initial experiments using standard differential centrifugation methods, they discovered that only a "small minority" of the extracellular miRNA content of the plasma samples was associated with the exosomal fraction. Still, it was possible that individual exosomes may carry significant numbers of miRNAs, the scientists wrote in PNAS.
To test this, the team turned to quantitative methods to directly determine the number of miRNAs of a given sequence contained within individual exosomes from both cancer patient plasma and other, diverse biological sources. These included different human biofluids like seminal fluid and plasma from healthy individuals, as well as human dendritic cells, ovarian cancer cells, and mast cells.
In all of the samples examined, the researchers found that the ratio of miRNA molecules for a given miRNA to the number of individual exosomes was substantially lower than one. Indeed, across all samples, they observed an average of one copy of miRNA detected per 121 exosomes, ranging from one copy per nine exosomes in the case of miR-720 in seminal fluid exosomes, to one copy per 47,162 exosomes in the case of miR-126 in healthy donor plasma exosomes.
"We found this result surprising, because it implied that most exosomes would not contain any copies of abundant miRNAs," the investigators noted in their paper.
In order to exclude the possibility that technical variables may have confounded their results, leading to an underestimation of miRNA abundance or an overestimation of exosome numbers, the scientists validated their quantification methods using orthogonal approaches. However, they were able to confirm empirically that a difference in neither PCR amplification efficiency nor exosome quantification could explain the results. They further eliminated the possibility that freezing and thawing of samples could have skewed their findings.
Though the findings may complicate existing mechanistic models of exosome-mediated miRNA transport, Tewari noted that they don't rule them out entirely. Rather, "the results mean that there are some models that are still possible and there are some models that are unlikely," he said.
In light of the new data, it is unlikely is that all exosomes will have a moderate-to-high copy number of miRNAs, he added. "What is possible is that there are rare exosomes that have a lot of microRNAs … and these may be the bioactive ones. Alternatively, exosome miRNAs may be acting by more potent, non-canonical mechanisms that are not yet fully understood."