Phosphatidylserine-Exposing Annexin A1-Positive Extracellular Vesicles: Potential Cancer Biomarkers

Area of Science:

• Extracellular Vesicle Biology
• Cancer Biomarkers
• Immunology

Background:

• Phosphatidylserine (PS) typically resides on the inner cell membrane but externalizes during apoptosis as an 'eat-me' signal.
• Viable cells, including tumor cells, can expose PS on their surface, particularly on extracellular vesicles (EVs).
• PS-exposing EVs are proposed cancer biomarkers, but their subtypes and detection require further study.

Purpose of the Study:

• To investigate and compare phosphatidylserine (PS) externalization on small extracellular vesicles (sEVs) and medium/large extracellular vesicles (m/lEVs) derived from breast cancer and non-cancerous cells.
• To evaluate the efficacy of annexin A5 and Protein S (GlaS) as PS-binding probes for EV analysis.
• To identify potential EV subtypes indicative of cancer through PS exposure analysis.

Main Methods:

• Enrichment of sEVs and m/lEVs from conditioned media of MDA-MB-231, MDA-MB-468, keratinocyte, and fibroblast cell lines.
• Analysis of PS externalization using a bead-based EV assay combined with flow cytometry.
• Comparison of PS detection using recombinant annexin A5 and carboxylated glutamic acid domain of Protein S (GlaS).
• Single EV flow cytometry to analyze PS externalization on individual sEVs and m/lEVs.

Main Results:

• Bulk EV assays indicated higher PS externalization in m/lEVs from MDA-MB-468 cells, with varying results for MDA-MB-231 cells.
• Higher GlaS binding was observed in m/lEVs from fibroblasts compared to cancer cells in bulk assays.
• Single EV flow cytometry revealed significantly higher PS externalization in cancer cell-derived m/lEVs (annexin A1+) compared to non-cancerous cells.
• Annexin A1 positivity was used to identify specific m/lEV subtypes in the analysis.

Conclusions:

• Phosphatidylserine (PS)-exposing medium/large extracellular vesicles (m/lEVs), specifically annexin A1-positive subtypes, are significantly elevated in cancer cells.
• These PS-exposing m/lEVs represent a promising, yet undervalued, EV subtype for early cancer detection.
• Further understanding of PS externalization on disease-associated EV subtypes can enhance diagnostic strategies.