Multiplexed exosome analyses with DNA barcoding

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 DESCRIPTION (provided by applicant): Circulating exosomes have emerged as a new class of biomarker which enables non- invasive, real-time disease monitoring. Most cancer cells actively release large numbers of exosomes into the circulation, that carry molecular constituents of the originating cells. Capturing such information can thus represent a new avenue to probe and serially monitor the tumor molecular status. We have previously developed miniaturized platforms to facilitate exosome analyses and established the clinical utility of exosomes for cancer diagnosis and monitoring through subsequent clinical studies. Predicting and detecting the emergence of drug resistance, however, is still challenging, as it requires multifaceted profiling of exosomal proteins, their post-translational modifications, and mRNA changes. The overall goal of this application is to advance a new screening technology for comprehensive exosomal protein/mRNA profiling. We will specifically explore the DNA-barcode labeling system to unify protein and mRNA detection into a single assay format: antibodies will be labeled with DNA tags whose sequences are unique for different protein targets; and ligation-dependent DNA tags will be used to detect mRNA targets. In Aim 1, we will develop and validate the proposed assay. We will implement a new, integrated fluidic system to perform cancer-specific exosome enrichment and DNA-barcoding on-chip. The device will be fabricated in thermoplastics (via injection molding) to promote system robustness and scaled-up production. In Aim 2, we will investigate the utility of exosomal protein/mRNA profiling in predicting and monitoring drug resistance in vitro and in vivo. The developed platform will be applied to analyze exosomes collected from ovarian cell lines and ovarian cancer patients undergoing therapies. Exosomal protein and mRNA targets will be monitored at baseline and longitudinally to differentiate treatment response and monitor the emergence of drug resistance. We envision that the new assay technology would have significant clinical implications by accelerating the translation of exosomes, not only as a cancer diagnostic biomarker but also as an indicator of drug efficacy and as a potential molecular stratifier for treatment decision.