Extracellular vesicles (EVs) provide an invaluable tool to analyse physiological processes because they transport, in biological fluids, biomolecules secreted from diverse tissues of an individual. EV biomarker detection requires highly sensitive techniques able to identify individual molecules. However, the lack of widespread, affordable methodologies for high-throughput EV analyses means that studies on biomarkers have not been done in large patient cohorts. To develop tools for EV analysis in biological samples, we evaluated here the critical parameters to optimise an assay based on immunocapture of EVs followed by flow cytometry. We describe a straightforward method for EV detection using general EV markers like the tetraspanins CD9, CD63 and CD81, that allowed highly sensitive detection of urinary EVs without prior enrichment. In proof-of-concept experiments, an epithelial marker enriched in carcinoma cells, EpCAM, was identified in EVs from cell lines and directly in urine samples. However, whereas EVs isolated from 5–10 ml of urine were required for western blot detection of EpCAM, only 500 μl of urine were sufficient to visualise EpCAM expression by flow cytometry. This method has the potential to allow any laboratory with access to conventional flow cytometry to identify surface markers on EVs, even non-abundant proteins, using minimally processed biological samples.

Therapy of metastatic melanoma advanced recently with the clinical implementation of signalling pathway inhibitors, such as vemurafenib, specifically targeting mutant BRAF. In general, patients experience remarkable clinical responses under BRAF inhibitor (BRAFi) treatment but eventually progress within 6-8 months due to resistance development. Responding metastases show an increased immune cell infiltrate, including also NK cells, that, however, is no longer detectable in BRAFi-resistant lesions, suggesting NK cell activity should be exploited to prevent disease progression. Here, we examined the effects of BRAFi on the expression of ligands targeting activating NK cells receptors immediately after treatment onset, prior to resistance development. We demonstrate that BRAF mutant melanoma cells cultured in the presence of vemurafenib, strongly decreased surface expression of ligands for NK activating receptors including the NKG2D-ligand, MICA, and the DNAM-1 ligand, CD155, and became significantly less susceptible to NK cell attack. NKG2D-ligand protein downregulation was due to a significant decrease in mRNA levels, already detectable 24 h after drug treatment. Interestingly, vemurafenib-induced MICA downregulation could be counteracted by treatment of melanoma cells with the histone deacetylase (HDAC) inhibitor (HDACi) sodium butyrate, that also upregulated the DNAM1-ligand, Nectin-2. HDACi treatment enhanced surface expression of NKG2D-ligands in the presence of BRAFi, accompanied by recovery of NK cell recognition, but only upon simultaneous drug application. These results suggest that co-administration of BRAFi and HDAC inhibitors as well as having direct effects on melanoma cell survival, could also synergise to improve NK cell recognition and avoid tumour immune evasion.

BackgroundTumour-derived exosomes can be released to serum and provide information on the features of the malignancy, however, in order to perform systematic studies in biological samples, faster diagnostic techniques are needed, especially for detection of low abundance proteins. Most human cancer cells are positive for at least one ligand for the activating immune receptor NKG2D and the presence in plasma of NKG2D-ligands can be associated with prognosis.MethodsUsing MICA as example of a tumour-derived antigen, endogenously expressed in metastatic melanoma and recruited to exosomes, we have developed two immunocapture-based assays for detection of different epitopes in nanovesicles. Although both techniques, enzyme-linked immunosorbent assay (ELISA) and Lateral flow immunoassays (LFIA) have the same theoretical basis, that is, using capture and detection antibodies for a colorimetric read-out, analysis of exosome-bound proteins poses methodological problems that do not occur when these techniques are used for detection of soluble molecules, due to the presence of multiple epitopes on the vesicle.ResultsHere we demonstrate that, in ELISA, the signal obtained was directly proportional to the amount of epitopes per exosome. In LFIA, the amount of detection antibody immobilized in Au-nanoparticles needs to be low for efficient detection, otherwise steric hindrance results in lower signal. We describe the conditions for detection of MICA in exosomes and prove, for the first time using both techniques, the co-existence in one vesicle of exosomal markers (the tetraspanins CD9, CD63 and CD81) and an endogenously expressed tumour-derived antigen. The study also reveals that scarce proteins can be used as targets for detection antibody in LFIA with a better result than very abundant proteins and that the conditions can be optimized for detection of the protein in plasma.ConclusionsThese results open the possibility of analyzing biological samples for the presence of tumour-derived exosomes using high throughput techniques.Electronic supplementary materialThe online version of this article (10.1186/s12951-018-0372-z) contains supplementary material, which is available to authorized users.

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Communication within the immune system depends on the release of factors that can travel and transmit information at points distant from the cell that produced them. In general, immune cells use two key strategies that can occur either at the plasma membrane or in intracellular compartments to produce such factors, vesicle release and proteolytic cleavage. Release of soluble factors in exosomes, a subset of vesicles that originate from intracellular compartments, depends generally on biochemical and lipid environment features. This physical environment allows proteins to be recruited to membrane microdomains that will be later endocytosed and further released to the extracellular milieu. Cholesterol and sphingolipid rich domains (also known as lipid rafts or detergent-resistant membranes, DRMs) often contribute to exosomes and these membrane regions are rich in proteins modified with Glycosyl-Phosphatidyl-Inositol (GPI) and lipids. For this reason, many palmitoylated and GPI-anchored proteins are preferentially recruited to exosomes. In this review, we analyse the biochemical features involved in the release of NKG2D-ligands as an example of functionally related gene families encoding both transmembrane and GPI-anchored proteins that can be released either by proteolysis or in exosomes, and modulate the intensity of the immune response. The immune receptor NKG2D is present in all human Natural Killer and T cells and plays an important role in the first barrier of defense against tumor and infection. However, tumor cells can evade the immune system by releasing NKG2D-ligands to induce down-regulation of the receptor. Some NKG2D-ligands can be recruited to exosomes and potently modulate receptor expression and immune function, while others are more susceptible to metalloprotease cleavage and are shed as soluble molecules. Strikingly, metalloprotease inhibition is sufficient to drive the accumulation in exosomes of ligands otherwise released by metalloprotease cleavage. In consequence, NKG2D-ligands appear as different entities in different cells, depending on cellular metabolism and biochemical structure, which mediate different intensities of immune modulation. We discuss whether similar mechanisms, depending on an interplay between metalloprotease cleavage and exosome release, could be a more general feature regulating the composition of exosomes released from human cells.