Researchers at Vanderbilt University Medical Center have discovered a nanoparticle released from cells, called a “supermere,” which contains enzymes, proteins and RNA associated with multiple cancers, cardiovascular disease, Alzheimer’s disease and even COVID-19.
The discovery, reported in Nature Cell Biology, is a significant advance in understanding the role extracellular vesicles and nanoparticles play in shuttling important chemical “messages” between cells, both in health and disease.
“We’ve identified a number of biomarkers and therapeutic targets in cancer and potentially in a number of other disease states that are cargo in these supermeres,” said the paper’s senior author, Robert Coffey, MD. “What is left to do now is to figure out how these things get released.”
Coffey, the Ingram Professor of Cancer Research and professor of Medicine and Cell & Developmental Biology, is internationally known for his studies of colorectal cancer. His team is currently exploring whether the detection and targeting of cancer-specific nanoparticles in the bloodstream could lead to earlier diagnoses and more effective treatment.
In 2019 Dennis Jeppesen, Ph.D., a former research fellow in Coffey’s lab who is now a research instructor in Medicine, used advanced techniques to isolate and analyze small membrane-enclosed extracellular vesicles called “exosomes.”
That year, using high-speed ultracentrifugation, another of Coffey’s colleagues, Qin Zhang, Ph.D., research assistant professor of Medicine, devised a simple method to isolate a nanoparticle called an “exomere” that lacks a surface coat.
In the current study, Zhang took the “supernatant,” or fluid that remains after the exomeres have been spun into a “pellet,” and spun the fluid faster and longer.
The result was a pellet of nanoparticles isolated from the supernatant of the exomere spin—which the researchers named supermeres. “They’re also super-interesting,” Coffey quipped, “because they contain many cargo previously thought to be in exosomes.”
For one thing, supermeres carry most of the extracellular RNA released by cells and which is found in the bloodstream. Among other functional properties, cancer-derived supermeres can “transfer” drug resistance to tumor cells, perhaps via the RNA cargo they deliver, the researchers reported.
Supermeres are important carriers of TGFBI, a protein that in established tumors promotes tumor progression. TGFBI thus may be a useful marker in liquid biopsies for patients with colorectal cancer, the researchers noted.
They also carry ACE2, a cell-surface receptor that plays a role in cardiovascular disease and is the target of the COVID-19 virus. This raises the possibility that ACE2 carried by supermeres could serve as a “decoy” to bind the virus and prevent infection.
Another potentially important cargo is APP, the amyloid-beta precursor protein implicated in the development of Alzheimer’s disease. Supermeres can cross the blood-brain barrier, suggesting that their analysis could improve early diagnosis or possibly even targeted treatment of the disease.
“The identification of this rich plethora of bioactive molecules … raises interesting questions about the function of supermeres, and heightens interest in the potential of these particles as biomarkers for diseases,” researchers at the University of Notre Dame noted in a review published with the paper.
Heterogeneity of EVs and nanoparticle populations is a major challenge in the EV field1,2,3,4,5. Here we report the isolation and characterization of a distinct extracellular nanoparticle that we have termed supermere. Supermeres are distinct from exomeres in terms of size, morphology, composition, cellular-uptake dynamics and tissue distribution. Our current AFM and electron microscopy data do not allow us to define structural differences between exomeres and supermeres beyond the diameter, height and volume differences identified by fluid-phase AFM.
Efforts are ongoing to examine these nanoparticles by cryogenic electron microscopy to determine their structural differences more precisely. In tissue biodistribution experiments, we consistently found greater final uptake of supermeres in vivo compared with sEVs, despite the slower uptake kinetics of supermeres (and exomeres) in vitro. An explanation for this discrepancy may reflect important differences in how cells interact with nanoparticles versus sEVs or merely be due to the technical limitations of our experiments. Future studies will be needed to resolve this issue.
Supermeres contain many proteins that have previously been reported to be associated with exosomes5. For example, TGFBI, the most abundant protein in supermeres, is purportedly a component of EVs from mesenchymal stromal cells51. Based on our findings that patients with CRC whose tumours exhibit high TGFBI immunoreactivity have a poor outcome and that the levels of TGFBI, determined by ELISA, are markedly increased in sEV-Ps, exomeres and supermeres isolated from the plasma of patients with CRC compared with those isolated from control individuals, we propose that TGFBI may be a useful marker in liquid biopsies for patients with CRC. TGFBI has been linked to both cancer cell migration52 and an immunosuppressive tumour microenvironment53. TGFBI mediates binding to extracellular-matrix proteins such as collagen and fibronectin, and can interact with integrin proteins52,53.
Future studies will focus on whether TGFBI-associated supermeres are responsible for these effects. Argonaute proteins, including AGO1 and AGO2, were presumed exosomal proteins but refinements in purification demonstrate that these miRNA-binding proteins are predominantly NV1,35 and associated with supermeres. Other known RNA-binding proteins are also enriched in supermeres, highlighting that a significant proportion of exRNAs and RNA-binding proteins are not associated with EVs1,54. Many miRNAs that are barely detectable or undetectable at the cellular level are highly and selectively enriched in supermeres. For example, miR-1246, which has been linked to serum exosomes in patients with CRC37, is the most highly expressed and highly enriched miRNA in supermeres. The strong staining of miR-1246 in CRC tissue compared with normal colonic mucosa supports miR-1246 as a biomarker with potential roles in the pathogenesis of CRC.
It is important to note that supermeres and exomeres are not the only NV extracellular nanoparticles capable of transporting miRNA as high-density lipoprotein (HDL) particles in plasma and serum are known to contain miRNA55,56. All the cell line-derived supermere samples generated for this work were from serum-free conditions with no detection of ApoA1 or ApoA2 (the most abundant proteins of HDL complexes) by proteomic analysis. However, efficient purification from HDL-rich blood may benefit from additional approaches, perhaps utilizing a combination of high-resolution density gradient fractionation1 and fast protein liquid chromatography or size-exclusion chromatography55,56 for improved separation of sEVs, exomeres, supermeres and HDL particles.
We demonstrated that supermeres and exomeres isolated from cetuximab-resistant SC and CC-CR cells can transfer cetuximab resistance to cetuximab-sensitive cells. Activation of the receptor tyrosine kinases MET and RON induce de novo cetuximab resistance in SC cells7. In CC-CR cells, upregulation of a long noncoding RNA (lncRNA), MIR100HG and two embedded miRNAs (miR-100 and miR-125b) is responsible for this acquired mode of cetuximab resistance16. Thus, multiple cargos, including proteins and RNA (messenger RNA, miRNA and lncRNA) carried by nanoparticles may contribute to these modes of drug resistance. The identity of these cargos, and whether they act independently or cooperatively in cetuximab resistance, await further investigation.
The Warburg effect features enhanced lactate secretion, acidification of the tumour microenvironment and extracellular-matrix degradation15. Lactate secretion has been linked to resistance to drugs targeting EGFR and MET17. We demonstrated that cancer cell-derived supermeres contain large amounts of glycolytic enzymes and their addition to recipient cells increases lactate secretion. Furthermore, treating mice with supermeres reduces the levels of lipids and glycogen in the liver. The liver phenotype we observed is similar to that reported with hepatic mTORC1 inhibition in which there was decreased hepatic steatosis and an increased inflammatory response44. Future studies will be needed to assign these effects on the liver to specific cargo in supermeres and exomeres.
Shedding or release of membrane receptors to the extracellular environment is associated with a number of disease states57 and drug resistance58. Secretion of full-length transmembrane receptors is, as we demonstrated, a distinctive feature of sEVs/exosomes but the ectodomain of many clinically relevant transmembrane receptors—including MET, GPC1, CEA, ACE, ACE2 and APP—are highly abundant in supermeres. As an example, the secreted receptor ACE2 in sEVs and extracellular nanoparticles may act as a decoy for SARS-CoV-2 to attenuate infection, as has been demonstrated for human soluble recombinant ACE2 (refs. 11,59). A GPI-anchor attached to the C terminus of a protein enables it to be attached to the plasma membrane of cells or EVs, and many GPI-anchored proteins of clinical importance—including GPC1, CEA, DPEP1 and CD73—have been detected in the extracellular space and ascribed to exosomes. However, GPC1 is less associated with exosomes, or other sEVs, but is instead enriched in exomeres and supermeres. Other GPI-anchored proteins (for example, DPEP1 and CD73) are strongly associated with EGFR+CD81+ exosomes. DPEP1 was recently identified as a neutrophil-binding receptor and targeting DPEP1 reduced mortality in murine models of sepsis, suggesting a role for DPEP1 in inflammation46. Here we demonstrated that increased diffuse DPEP1 staining is associated with overall and progression-free survival in CRC and increased levels of DPEP1+CEA+ exosomes are present in the plasma of patients with CRC. High levels of CD73 have been linked to immune suppression and tumour progression due to the generation of extracellular adenosine60. We found increased levels of CD73 in CRC tumour tissue and demonstrated that CD73+ exosomes can be detected in the plasma of patients with CRC.
Based on our findings, we propose that TGFBI, ENO1 and GPC1 may be useful markers for extracellular nanoparticles (exomeres and supermeres), whereas HSPA13 and ENO2 are more specifically associated with supermeres. Going forward, it will be important to elucidate the biogenesis of supermeres and exomeres. The abundance of retromer machinery associated with both of these amembranous nanoparticles may offer a clue. Equally important will be to unravel the mechanism(s) underlying the effects mediated by these extracellular nanoparticles and their cargo.
In summary, we have identified a distinct circulating extracellular nanoparticle. Supermeres are enriched in proteins and miRNAs central to a number of disease states, including cancer, COVID-19, cardiovascular disease and Alzheimer’s disease. Many of these proteins have previously been ascribed to exosomes or other sEVs. Our findings serve to highlight the importance of parsing the exact extracellular compartment that contains a biomolecule of interest. Supermeres are also functional agents of intercellular communication that are efficiently taken up by multiple organs, including the liver, lung, colon, heart and brain. Thus, supermeres take their place alongside EVs and exomeres as a rich source of circulating cargo for candidate biomarkers and therapeutic targets in a number of disease states.
More information: Qin Zhang et al, Supermeres are functional extracellular nanoparticles replete with disease biomarkers and therapeutic targets, Nature Cell Biology (2021). DOI: 10.1038/s41556-021-00805-8