Development of a multiplex-to-single exosome analysis (MT-SEA) pipeline to characterize exosomes associated with tumour progression and responses to treatment

Background: Extracellular vesicles (EVs) have potential as non-invasive biomarkers. We developed a first-in-class pipeline to characterize EV heterogeneity and provide high-sensitivity quantification of informative EVs in biofluids throughout treatment. By combining multiplex assays with high-resolution, single EV flow cytometric methods together into a mutiplex-to-single EV analysis (Mt-SEA) pipeline, we are able to characterize a broad range of EV subsets, while also measuring the concentration of specific EV populations. Exploratory studies presented here validate the Mt-SEA method by confirming strong correlations of liquid biopsy EV repertoires with tumour burden and responses to treatment. Methods: Plasma was obtained before and after treatment (n = 5 treatment courses) from Adult T-cell leukemia/lymphoma patients receiving palliative radiation. Multiplex EV capture beads were used with additional detection antibodies to identify 37 major EV subsets. General exosome and EV detection epitopes included CD63, CD9 and CD81. Tumour-specific epitopes for each patient included CD4, CD5 and CD25, based on available histo-/cyto-pathology results. High-resolution single EV analyses were performed with nanoFACS sorting and a prototype nanoFCM analyser. Results: ATLL-derived EVs were detected in each pretreatment sample, with reduced specific ATLL-derived EV subsets concentrations at the end of treatment. Furthermore, ATLL-specific EVs from patients with progressive systemic disease prior to treatment were found to carry CD44 and other stemness-associated epitopes, consistent with increasing tumour aggressiveness. Responses to treatment that were clinically evident mirrored changes in the Mt-SEA EV profiles, and Mt-SEA identified new candidate prognostic EV profiles associated with clinical outcomes. Summary/conclusion: Our exploratory study demonstrates that Mt-SEA provides unexpected insights into tumour biology, along with robust estimations of concentrations of EV subsets of interest. Detection of tumour-associated EVs and detection of EV repertoire changes during treatment paves the way to future evaluation of the Mt-SEA pipeline for personalized therapies in a wider range of tumour types.