Characterizing Glycosylated Cargo of Extracellular Vesicles in Luminal Breast Cancer
Abstract
Introduction: In the USA, one in eight women will be diagnosed with breast cancer sometime in their lifetime. In cancer biology, breast cancer is a heterogeneous and evolving disease. Unfortunately, the current standard of diagnosis cannot adequately recapitulate the inherent nature of breast cancer. Inadequate diagnostics often lead to suboptimal treatment, which could have grim consequences. Clinicians need a precision diagnostic technique that can reflect the biology of a patient's individual cancer. Extracellular vesicles (EVs), as the means of intercellular communication, hold the great potential for this purpose. Like their parent cells, EVs are highly glycosylated. However, little is known about the molecular basis of glycosylated EV cargos and their roles in breast cancer. Understanding EVs and their unique glycosylation patterns could provide a basis for a holistic EV-based diagnostic modality: EV-based liquid biopsy.
Methods: A luminal breast cancer model which includes a non-tumorigenic luminal epithelial cell line, MCF-10A, and three luminal breast cancer cell lines, MCF-7, LCC9, and BT474, were used in this study. EVs (including small extracellular vesicles (sEVs) and microvesicles (MVs)) were isolated from conditioned media by a differential ultracentrifugation approach. The quality and quantity of EVs were assessed by transmission electron microscopy, NanoSight, and western blotting with a panel of markers. Metabolic labeling (with ManNAz and GalNAz) and click chemistry were employed to characterize cellular and EV surface glycoproteins.
Results: Enriched particles with nanoscale size (sEV: 30-200 nm, MVs: 100-1000 nm) harbored membrane-encapsulated vesicular structure and presented typical EV markers, which are consistent with previous literature. On average, MCF10A produces the least sEVs per cell while MCF7 produces the most. There is no significant difference in MV production across all four cell lines. Abundant glycoproteins were detected on the surface of cells and sEVs with specific labeled signals. Glycosylation patterns and levels on the EV surface varied across different breast cancer cell lines.
Conclusion: These results provide the foundation towards building sEV and MV "fingerprints" that are specific to each cell line and ergo the breast cancer subtypes.