A Single-Step Immunocapture Assay to Quantify HCC Exosomes Using the Highly Sensitive Fluorescence...

Area of Science:

• Biomarkers
• Cancer Research
• Nanotechnology

Background:

• Extracellular vesicles (EVs) show potential as non-invasive biomarkers for early cancer detection.
• Quantifying cancer-derived EVs in native states is challenging for clinical translation.

Purpose of the Study:

• To develop a rapid, one-step immunoaffinity assay for quantifying hepatocellular carcinoma (HCC)-derived exosomes in small serum volumes.

Main Methods:

• Developed a fluorescence nanoparticle tracking analysis (F-NTA) assay using phycoerythrin (PE)-conjugated antibodies targeting GPC3 and alpha-fetoprotein (AFP) for HCC exosome capture.
• Quantified total and HCC-specific exosomes in serum and correlated results with tumor size determined by MRI.

Main Results:

• The F-NTA assay demonstrated sensitivity with a detection range of 10^4-10^8 particles/mL.
• HCC cell cultures released significantly higher quantities of GPC3 or GPC3+AFP positive EVs compared to non-HCC cultures.
• The assay showed selective enrichment of AFP and GPC3 positive EVs in HCC serum (AUC values 0.79 for GPC3, 0.71 for AFP, 0.72 for GPC3/AFP).
• A positive correlation was observed between tumor size and GPC3-positive exosome concentration (r=0.78, p<0.001).

Conclusions:

• An immunocapture assay was successfully developed for simultaneous isolation and quantification of HCC-derived exosomes from small serum volumes.
• The assay provides high accuracy and preserves the biological immunoreactivity of exosomes.
• This method holds promise for accurate, non-invasive early detection of HCC.