Digital Profiling of Tumor Extracellular Vesicle-Associated RNAs Directly from Unprocessed Blood Plasma

Area of Science:

• Biomarker Discovery
• Molecular Diagnostics
• Extracellular Vesicle Research

Background:

• Tumor-derived extracellular vesicles (tEVs) contain RNA biomarkers with diagnostic potential.
• The clinical utility of tEV RNAs is limited by their low abundance among non-tumor EVs.
• Sensitive methods are needed to detect and profile RNA from rare tEVs.

Purpose of the Study:

• To develop a highly sensitive digital method for profiling RNA from extracellular vesicles (EVs).
• To enable precise quantification of rare EV subpopulations for biomarker discovery.
• To overcome challenges in detecting tumor-derived biomarkers in liquid biopsies.

Main Methods:

• EV-CLIP, a droplet-based digital method, fuses EVs with charged liposomes (CLIPs) in a microfluidic chip.
• Optimized CLIP surface charge enhances sensitivity and selectivity for EV-derived miRNAs and mRNAs.
• The method requires minimal plasma volume (20 μL) and bypasses EV isolation and RNA preparation steps.

Main Results:

• EV-CLIP demonstrated exceptional sensitivity and selectivity for EV RNA profiling.
• The method successfully detected EGFR mutations (L858R and T790M) in 83 patient samples with high AUC values (1.0000 and 0.9784).
• Serial monitoring during chemotherapy showed the method's capability for precise quantification of rare EV subpopulations.

Conclusions:

• EV-CLIP offers a sensitive and streamlined approach for profiling EV RNA from minimal plasma volumes.
• This technology facilitates the detection of rare tumor-derived biomarkers and supports precise monitoring of cancer therapy.
• EV-CLIP has the potential to advance the understanding of EV heterogeneity in various disease states and improve clinical diagnostics.