Assessing Breast Cancer Molecular Subtypes Using Extracellular Vesicles’ mRNA
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Issue Date
2023Author
Hu, Mengjia
Brown, Virginia
Jackson, Joshua M.
Wijerathne, Harshani
Pathak, Harsh
Koestler, Devin C.
Nissen, Emily
Hupert, Mateusz L.
Muller, Rolf
Godwin, Andrew K.
Witek, Malgorzata A.
Soper, Steven A.
Publisher
American Chemical Society
Type
Article
Article Version
Scholarly/refereed, author accepted manuscript
Rights
Copyright © 2023 American Chemical Society
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Show full item recordAbstract
Extracellular vesicles (EVs) carry RNA cargo that is believed to be associated with the cell-of-origin and thus have the potential to serve as a minimally invasive liquid biopsy marker for supplying molecular information to guide treatment decisions (i.e., precision medicine). We report the affinity isolation of EV subpopulations with monoclonal antibodies attached to the surface of a microfluidic chip that is made from a plastic to allow for high-scale production. The EV microfluidic affinity purification (EV-MAP) chip was used for the isolation of EVs sourced from two-orthogonal cell types and was demonstrated for its utility in a proof-of-concept application to provide molecular subtyping information for breast cancer patients. The orthogonal selection process better recapitulated the epithelial tumor microenvironment by isolating two subpopulations of EVs: EVEpCAM (epithelial cell adhesion molecule, epithelial origin) and EVFAPα (fibroblast activation protein α, mesenchymal origin). The EV-MAP provided recovery >80% with a specificity of 99 ± 1% based on exosomal mRNA (exo-mRNA) and real time–droplet digital polymerase chain reaction results. When selected from the plasma of healthy donors and breast cancer patients, EVs did not differ in size or total RNA mass for both markers. On average, 0.5 mL of plasma from breast cancer patients yielded ∼2.25 ng of total RNA for both EVEpCAM and EVFAPα, while in the case of cancer-free individuals, it yielded 0.8 and 1.25 ng of total RNA from EVEpCAM and EVFAPα, respectively. To assess the potential of these two EV subpopulations to provide molecular information for prognostication, we performed the PAM50 test (Prosigna) on exo-mRNA harvested from each EV subpopulation. Results suggested that EVEpCAM and EVFAPα exo-mRNA profiling using subsets of the PAM50 genes and a novel algorithm (i.e., exo-PAM50) generated 100% concordance with the tumor tissue.
Description
This document is the Accepted Manuscript version of a Published Work that appeared in final form in Analytical Chemistry, copyright © 2023 American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acs.analchem.3c00624.
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Citation
Hu, M., Brown, V., Jackson, J. M., Wijerathne, H., Pathak, H., Koestler, D. C., Nissen, E., Hupert, M. L., Muller, R., Godwin, A. K., Witek, M. A., & Soper, S. A. (2023). Assessing Breast Cancer Molecular Subtypes Using Extracellular Vesicles' mRNA. Analytical chemistry, 95(19), 7665–7675. https://doi.org/10.1021/acs.analchem.3c00624
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