Immunoassays for scarce tumour-antigens in exosomes: detection of the human NKG2D-Ligand, MICA, in tetraspanin-containing nanovesicles from melanoma

Abstract

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.

MICAgen Mice Recapitulate the Highly Restricted but Activation-Inducible Expression of the Paradigmatic Human NKG2D Ligand MICA

“…Further, our data suggest that sMICA in plasma of MICAgen mice is, at least in part, derived from hematopoietic cells that up-regulated and shed MICA upon activation ( Figure 5). Such sMICA may differ in its effects on NKG2D modulation from tumor-released MICA, especially when embedded in exosomes (45,48,49). Of note, activation-induced surface MICA is vanishing over several days past activation, and it will be interesting to determine the mechanisms not only underlying MICA induction but also underlying the reversal of MICA surface expression.…”

Extracellular Vesicles as Biomarkers in Cancer Immunotherapy

“…TDE expressing NKG2D ligands are a means that tumor cells use for immune-evasion [ 74 ]. It has been demonstrated that ovarian cancer and melanoma TDEs, express NKG2D ligands and prevent activation of cytotoxic NK cells [ 75 , 76 ]. Expression of FASL and TRAIL on TDEs induce apoptosis in dendritic cells (DCs) and peripheral blood mononuclear cells (PBMCs) which causes immunosuppression and promotes tumor progression [ 77 ].…”

The Angiogenic Paracrine Potential of Mesenchymal Stem Cells

“…The feasibility of exosome uptake by recipient cells, make these cell products for introducing in clinical approaches. Xue and colleagues investigated the effects of cord blood and adipose-derived MSC exosomes on human EC angiogenesis capacity under hypoxic and normal conditions [106,107]. They noted the potency of isolated exosomes in triggering angiogenesis rate especially under the hypoxic condition compared to exosome counterpart originated from normal milieu.…”