Abstract 3978: Direct analysis of exosome-bound proteins and nucleic acids using the ExoVerita™ Flex platform

Abstract

Abstract Exosomes carry signals between cells and may influence cancer development, metastasis, and drug resistance. Unfortunately, current tools for exosome isolation and analysis have been limited to examining a specific class of markers, such as DNA, RNA, and proteins. We have developed a microelectrode array chip that uses a novel Alternating Current Electrokinetic method to perform rapid, affinity-free isolation of exosomes. The chip runs on our proprietary ExoVeritaTM Flex platform. Isolated exosomes can then be analyzed in situ or can be lysed to obtain DNA and RNA for use in downstream assays, such as sequencing. In this study, we used the ExoVerita Flex to isolate exosomes directly from plasma specimens obtained from cancer subjects with lung, colorectal, or pancreatic cancers as well as healthy donors. Isolated exosomes were analyzed in situ for exosome-bound proteins and nucleic acids, and then purified following isolation to examine their nucleic acid expression profiles. Using the ExoVerita Flex with plasma specimens and antibodies targeting the exosome-bound proteins CEA, PD-L1, GPC-1 and CD63, we observed differences in protein expression levels among the different cancer types (N = 35) and healthy donors (N = 20), such as increased PD-L1 expression in non-small cell lung cancer subject samples and elevated levels of CEA in colorectal cancer subject samples (N = 10). Furthermore, we co-stained samples with antibodies and DNA- or RNA-selective dyes to simultaneously measure exosome-bound proteins and circulating nucleic acid levels in the same sample at the same time. RNA sequencing analysis was performed by extracting RNA from the isolated exosomes using a proprietary buffer, and then performing RT-ddPCR and miRNA-Sequencing. RT-ddPCR analysis of the PGK-1 housekeeping gene RNA transcript confirmed the presence of viable RNA following extraction. miRNA profiles from isolated exosomes were generated using a targeted RNA sequencing assay and used for repeatability and differential expression studies. The ExoVerita Flex platform offers a novel, unbiased method for isolation and characterization of exosome-bound proteins and circulating nucleic acids while being compatible with standard downstream analysis. The ability to perform two-dimensional protein & nucleic acid analysis in the same sample run, at the same time, is both unique and unprecedented and will enable promising future clinical studies in cancer looking at diverse classes of biomarkers directly from blood. Citation Format: Jean M. Lewis, David Searson, Alfred Kinana, Heath Balcer, Debrah Thompson, David Bodkin, Juan P. Hinestrosa, Rajaram Krishnan. Direct analysis of exosome-bound proteins and nucleic acids using the ExoVerita™ Flex platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3978.