Colocalization of Cancer-Associated Biomarkers on Single Extracellular Vesicles for Early Detection of Cancer.

Detection of cancer early, when it is most treatable, remains a significant challenge because of the lack of diagnostic methods sufficiently sensitive to detect nascent tumors. Early-stage tumors are small relative to their tissue of origin, heterogeneous, and infrequently manifest in clinical symptoms. Detection of their presence is made more difficult by a lack of abundant tumor-specific indicators (ie, protein biomarkers, circulating tumor DNA) that would enable detection using a noninvasive diagnostic assay. To overcome these obstacles, we have developed a liquid biopsy assay that interrogates circulating extracellular vesicles (EVs) to detect tumor-specific biomarkers colocalized on the surface of individual EVs. We demonstrate the technical feasibility of this approach in human cancer cell line-derived EVs, where we show strong correlations between assay signal and cell line gene/protein expression for the ovarian cancer-associated biomarkers bone marrow stromal antigen-2, folate receptor-α, and mucin-1. Furthermore, we demonstrate that detecting distinct colocalized biomarkers on the surface of EVs significantly improves discrimination performance relative to single biomarker measurements. Using this approach, we observe promising discrimination of high-grade serous ovarian cancer versus benign ovarian masses and healthy women in a proof-of-concept clinical study.

Peptide-based urinary monitoring of fibrotic nonalcoholic steatohepatitis by mass-barcoded activity-based sensors.

Noninvasive detection of nonalcoholic steatohepatitis (NASH), the progressive form of nonalcoholic fatty liver disease, promises to improve patient screening, accelerate drug trials, and reduce health care costs. On the basis of protease dysregulation of the biological pathways of fibrotic NASH, we developed the Glympse Bio Test System (GBTS) for multiplexed quantification of liver protease activity. GBTS-NASH comprises a mixture of 19 mass-barcoded PEGylated peptides that is administered intravenously and senses liver protease activity by releasing mass-barcoded reporters into urine for analysis by mass spectrometry. To identify a protease signature of NASH, transcriptomic analysis of 355 human liver biopsies identified a 13-protease panel that discriminated clinically relevant NASH ≥F2 fibrosis from F0-F1 with high classification accuracy across two independent patient datasets. We screened 159 candidate substrates to identify a panel of 19 peptides that exhibited high activity for our 13-protease panel. In the choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) mouse model, binary classifiers trained on urine samples discriminated fibrotic NASH from simple steatosis and healthy controls across a range of nondisease conditions and indicated disease regression upon diet change [area under receiver operating characteristics (AUROCs) > 0.97]. Using a hepatoprotective triple combination treatment (FXR agonist, ACC and ASK1 inhibitors) in a rat model of NASH, urinary classification distinguished F0-F1 from ≥F2 animals and indicated therapeutic response as early as 1 week on treatment (AUROCs >0.91). Our results support GBTS-NASH to diagnose fibrotic NASH via an infusion of peptides, monitor changes in disease severity, and indicate early treatment response.

Induction of tumor-specific protective immunity by in situ Langerhans cell vaccine.

Although anti-tumor immunity is inducible by dendritic cell (DC)-based vaccines, time- and cost-consuming "customizing" processes required for ex vivo DC manipulation have hindered broader clinical applications of this concept. Epidermal Langerhans cells (LCs) migrate to draining lymph nodes and undergo maturational changes on exposure to reactive haptens. We entrapped these migratory LCs by subcutaneous implantation of ethylene-vinyl-acetate (EVA) polymer rods releasing macrophage inflammatory protein (MIP)-3beta (to create an artificial gradient of an LC-attracting chemokine) and topical application of hapten (to trigger LC emigration from epidermis). The entrapped LCs were antigen-loaded in situ by co-implantation of the second EVA rods releasing tumor-associated antigens (TAAs). Potent cytotoxic T-lymphocyte (CTL) activities and protective immunity against tumors were induced efficiently with each of three tested TAA preparations. Thus, tumor-specific immunity is inducible by the combination of LC entrapment and in situ LC loading technologies. Our new vaccine strategy requires no ex vivo DC manipulation and thus may provide time and cost savings.