Introduction: Extracellular vesicles (EVs) have garnered much recent attention as circulating biomarkers for non-invasive tumour monitoring 1. EVs are membranous nanoparticles which are secreted from a variety of cell types, including tumour cells. The clinical utility of EVs as circulating biomarkers is contingent on a thorough understanding of the correlation between tumour-EV cargo and the molecular features of the originating tumour cell. As such, the current study aimed to characterise the breast cancer EV proteome and evaluate if cellular features including ER and HER2 expression were reflected in the EV content.
Methods: EVs were isolated from the conditioned media of nine breast cancer cell lines using an optimised size exclusion chromatography protocol2. The isolated EVs were enumerated and physically sized using nanoparticle tracking analysis (NTA). Following this, label-free shotgun proteomics was performed using a nanoLC-MS/MS workflow, and proteins that were differentially abundant based on the ER and HER2 status of the originating cell were identified.
Results: NTA confirmed that nano-sized vesicles were recovered from all cell lines. The proteomics data suggested breast cancer cell-derived EVs shared core protein cargo, including established EV markers such as clathrin heavy chain 1, programmed cell death 6-interacting protein (ALIX) and CD9 antigen. Upregulated proteins in ER+ EVs included E-cadherin (CDH1), prominin-2 (PROM2) and mucin-5B (MUC5B). Unsurprisingly, HER2 protein was upregulated in HER2+ EVs, along with sushi domain-containing protein 2 (SUSD2) and mucin-6 (MUC6). A signature of 12 EV proteins was derived that appeared to recapitulate the ER and HER2 expression status of the originating cell.
Conclusion: This study represents an important foundational step in understanding the correlation between breast cancer cell and EV content. With further validation, the identified protein candidates may have utility as part of a liquid biopsy for characterising key molecular features of breast cancer.