Development of an Exosome / EV Analysis Pipeline for Tumor and Immune Monitoring

Authors

• JC Jones
• JA Welsh
• J Kepley
• TA Waldmann
• K Conlon
• G Franchini
• JL Gulley
• KA Camphausen
• JA Berzofsky

Abstract

Exosomes and other Extracellular Vesicles (EVs) carry surface receptors that are characteristic of their cells of origin. Therefore, Extracellular Vesicles (EVs) have tremendous potential as non-invasive biomarkers for immunotherapy, but the use of EVs as clinical biomarkers requires a combination of methods to broadly characterize EVs and rigorously enumerate specific selected EV subsets. We developed a first-in-class pipeline to characterize EV heterogeneity and provide high-sensitivity quantification of informative EVs in biofluids before, during, and after treatment. This pipeline combines multiplex assays with high-resolution single EV flow cytometric methods together into a Mutiplex-to-Single EV Analysis (Mt-SEA) pipeline. To evaluate the use of this pipeline in an exploratory clinical cohort, we evaluated EVs from plasma samples of Adult T Cell Leukemia/Lymphoma patients receiving palliative radiation. Plasma was obtained before and after treatment. Multiplex EV capture beads were used with additional detection antibodies to identify more than 40 major EV subsets. while also accurately measuring the concentration of specific EV populations. With clinical cases, we demonstrate the performance of Mt-SEA method by confirming strong correlations of liquid biopsy EV repertoires with tumor burden and responses to treatment, including an abscopal immune response following radiation. Furthermore, EV analysis with Mt-SEA may identify previously unrecognized prognostic epitopes or EVs subsets. Mt-SEA provides unexpected insights into tumor biology, and detection of tumor-associated and immune-associated EVs and detection of EV repertoire changes during treatment paves the way to future evaluation of the Mt-SEA pipeline for personalized, bio-adaptive therapies in a wider range of tumor types.

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