Analyzing Extracellular Vesicle-associated DNA Using Transmission Electron Microscopy at the Single EV-level

Area of Science:

• Cell Biology
• Biochemistry
• Biomarker Discovery

Background:

• Extracellular vesicles (EVs) mediate cell-cell communication via cargo transfer, including DNA.
• EV-associated DNA (EV-DNA) is a potential liquid biopsy biomarker but challenging to analyze due to EV nanoscale structure and low abundance.
• Accurate detection protocols for EV-DNA are crucial for understanding its physiological and pathological roles.

Purpose of the Study:

• To develop and validate novel protocols for the accurate detection and characterization of EV-DNA.
• To confirm the presence and localization of double-stranded DNA within extracellular vesicles.
• To establish methods for identifying EV-associated DNA biomarkers using advanced microscopy.

Main Methods:

• Protocol 1: Combines ultrathin sectioning of EVs with immunogold labeling and transmission electron microscopy (TEM) for detecting lumenal dsDNA.
• Protocol 2: Employs whole-mount EV immunogold labeling for morphological analysis and surface-associated DNA detection.
• Utilizes TEM imaging for high-resolution visualization of EV structure and DNA localization.

Main Results:

• Demonstrated simultaneous detection of dsDNA and the EV surface protein tetraspanin 9 on cancer-cell-derived EVs using TEM.
• Validated the presence of EV-DNA within the EV lumen and on the EV surface.
• Established a versatile method for labeling internal and surface EV proteins.

Conclusions:

• The developed protocols enhance the accurate detection and characterization of EV-DNA.
• These methods facilitate single EV analysis and the identification of EV-associated biomarkers.
• The findings support the potential of EV-DNA as a biomarker in liquid biopsy and disease diagnostics.