Exosome research has grown exponentially due to the recognition of the potential roles of exosomes in pathophysiological processes, including cancer. However, due to technical challenges, isolated nanovesicles constitute a heterogeneous population and this has hindered our understanding of their biogenesis, molecular composition, biodistribution, and functions in vivo, and has limited their translational application. The state-of- the-art technology, asymmetric-flow field-flow fractionation (AF4), exhibits unique capability to separate nanoparticles and has been widely utilized to characterize nanoparticles and polymers in the pharmaceutical industry as well as various biological macromolecules, protein complexes and viruses. The objective of this study is to develop and validate the application of AF4 in exosome isolation and identification of novel nanovesicles using pancreatic cancer as a model system. We will evaluate its application in analyzing exosomes isolated from a panel of established human pancreatic cancer cell lines (Aim 1). We will further develop and optimize the AF4 methodology to apply it to complex biological specimens such as blood plasma from human subjects (Aim 2). Lastly, we will validate the AF4 application for the fractionation and characterization of distinct exosome subpopulations and identification of other novel nanovesicles using specimens (blood plasma and tumor tissues) isolated from pancreatic patients with newly diagnosed disease and at different stages of disease as well as patients undergoing treatment (Aim 3). We predict that AF4 in combination with sensitive molecular assays can serve as an improved analytical tool for the isolation of specific nanovesicle subpopulations, thereby addressing the complexities of vesicle heterogeneity.

Public Health Relevance

The heterogeneity of extracellular vesicles and the technical limitations of efficient separation of these nanosized vesicles have hindered our understanding of their biogenesis, molecular composition, and functions in vivo and have limited their translational potential. We will develop and validate the application of a novel technology, asymmetric-flow field-flow fractionation (AF4), for the fractionation and characterization of distinct subpopulations of exosomes and identification of novel nanovesicles derived from pancreatic cancer cell lines as well as pancreatic cancer patient tumor explants and blood.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
3R01CA218513-02S1
Application #
9664270
Study Section
Cancer Biomarkers Study Section (CBSS)
Program Officer
Schwartz, Elena Ivan
Project Start
2017-07-15
Project End
2022-06-30
Budget Start
2018-07-01
Budget End
2019-06-30
Support Year
2
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Weill Medical College of Cornell University
Department
Pediatrics
Type
Schools of Medicine
DUNS #
060217502
City
New York
State
NY
Country
United States
Zip Code
10065
Zhang, Haiying; Freitas, Daniela; Kim, Han Sang et al. (2018) Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation. Nat Cell Biol 20:332-343