PROJECT SUMMARY The worldwide prevalence of obesity has reached pandemic proportions. An association between fat accumulation or hepatosteatosis and hepatocellular carcinoma (HCC) development has been long known. Obesity-induced hepatosteatosis, together with its more severe complication nonalcoholic steatohepatitis (NASH), classified as the nonalcoholic fatty liver disease (NAFLD) affects up to 40% of the US population. Based on correlative and bench studies, several mechanisms have been proposed to explain how obesity increase cancer risk. An important finding that accounts for the tumor-promoting effect of obesity is the low- grade, aberrant inflammatory response, which results in the elevated production of cytokines, such as TNF and IL-6. Studies with mouse models demonstrated that obesity-promoted HCC development is dependent on the enhanced production of these inflammatory cytokines. However, critical questions concerning how the aberrant inflammation in obesity is initiated have been yet to be clearly defined. We hypothesize that in obesity, extracellular vesicles (EVs) derived from hepatocytes become pathogenic and drive recipient immune cells, such as neutrophils and monocytes/macrophages, towards abnormal inflammation associated with the development of HCC. To demonstrate this hypothesis, we will establish two novel systems; A) a new mouse line in which specific cell/tissue-derived EVs are selectively labeled with green fluorescent protein (GFP), and B) a simple, yet powerful electrokinetic-based micro-device that can rapidly extract EVs based on their dielectric properties from biofluids with high purity and yield. With these systems, we aim to 1) determine if H-EVs become pathogenic and induce aberrant inflammation leading to HCC, and 2) isolate H-EVs through the novel dielectrophoretic (DEP) technology and assess their pro- inflammatory trait. This study will lead us to demonstrate the pathogenicity of hepatocyte-derived EVs, which potentially provides a novel mechanism for the development of HCC and the EV biomarker for the disease.

PROJECT NARRATIVE More than 90% of hepatocellular carcinoma (HCC) develops in the context of severe liver fibrosis and cirrhosis caused by chronic liver inflammation, and obesity has been established as a risk factor for HCC development. To determine the mechanisms by which obesity accelerates the HCC development, we will investigate the role of hepatocyte-derived extracellular vesicles (H-EVs) in the pathogenesis of HCC by establishing and utilizing two novel system; A) a new mouse line in which specific cell/tissue-derived EVs are selectively labeled with green fluorescent protein (GFP), and B) a simple, yet powerful electrokinetic-based micro-device that can rapidly extract EVs based on their dielectric properties from biofluids with high purity and yield. Through this proposed study, we aim to establish versatile and much-needed systems for cell-type- specific EV monitoring and rapid and reproducible EV isolation in order to demonstrate the pathogenicity of H-EVs in obesity, which potentially provides a novel mechanism for the development of HCC and the EV biomarker for the disease.