Because exosomes mirror the origin and status of the cells, the analysis of the content encapsulated in exosomes from biological fluids can reveal information relevant to human health and disease. However, due to the paucity of highly effective and rigorous exosome isolation methods, identifying exosome content carrying biological information remains challenging. To advance our understanding of exosomes from breast cancer cells, we developed an innovative exosome isolation technology. With this new tool, we will investigate the function of exosomes in early detection and staging of breast cancer. In Aim 1 we will expand our new isolation technology that reports exosome proteome and validate the functionality extensively. In Aim 2, we will investigate how the release of exosomes is modulated in breast cancer cells in in vitro cultures and cell-derived xenografts. Successful data collection and analysis from this proposed study will not only determine the applicability of this novel technique but will also uncover the association between exosomes and breast cancer cell states, thus providing molecular signatures that can potentially serve as biomarkers and therapeutic choices for breast cancer.

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 lowgrade, 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.