A Single-Step Immunocapture Assay to Quantify HCC Exosomes Using the Highly Sensitive Fluorescence Nanoparticle-Tracking Analysis

Ali Riza Koksal; Nergiz Ekmen; Yucel Aydin; Kelley Nunez; Tyler Sandow; Molly Delk; Martin Moehlen; Paul Thevenot; Ari Cohen; Srikanta Dash

doi:10.2147/JHC.S423043

A Single-Step Immunocapture Assay to Quantify HCC Exosomes Using the Highly Sensitive Fluorescence Nanoparticle-Tracking Analysis

J Hepatocell Carcinoma. 2023 Nov 2:10:1935-1954. doi: 10.2147/JHC.S423043. eCollection 2023.

Authors

Ali Riza Koksal¹, Nergiz Ekmen², Yucel Aydin², Kelley Nunez³, Tyler Sandow⁴, Molly Delk², Martin Moehlen², Paul Thevenot³, Ari Cohen^{3

5}, Srikanta Dash^{1

2

6}

Affiliations

¹ Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, New Orleans, LA, USA.
² Department of Gastroenterology and Hepatology, Tulane University Health Sciences Center, New Orleans, LA, USA.
³ Department of Gastroenterology and Hepatology, Institute of Translational Research, Ochsner Health, New Orleans, LA, USA.
⁴ Department of Radiology, Institute of Translational Research, Ochsner Health, New Orleans, LA, USA.
⁵ Multi-Organ Transplant Institute, Ochsner Health, New Orleans, LA, USA.
⁶ Southeast Louisiana Veterans Health Care System, New Orleans, LA, USA.

Abstract

Introduction: Extracellular vesicles could serve as a non-invasive biomarker for early cancer detection. However, limited methods to quantitate cancer-derived vesicles in the native state remain a significant barrier to clinical translation.

Aim: This research aims to develop a rapid, one-step immunoaffinity approach to quantify HCC exosomes directly from a small serum volume.

Methods: HCC-derived exosomes in the serum were captured using fluorescent phycoerythrin (PE)-conjugated antibodies targeted to GPC3 and alpha-fetoprotein (AFP). Total and HCC-specific exosomes were then quantified in culture supernatant or patient-derived serums using fluorescence nanoparticle tracking analysis (F-NTA). The performance of HCC exosome quantification in the serum was compared with the tumor size determined by MRI.

Results: Initially we tested the detection limits of the F-NTA using synthetic fluorescent and non-fluorescent beads. The assay showed an acceptable sensitivity with a detection range of 10⁴-10⁸ particles/mL. Additionally, the combination of immunocapture followed by size-exclusion column purification allows the isolation of smaller-size EVs and quantification by F-NTA. Our assay demonstrated that HCC cell culture releases a significantly higher quantity of GPC3 or GPC3+AFP positive EVs (100-200 particles/cell) compared to non-HCC culture (10-40 particles/cell) (p<0.01 and p<0.05 respectively). The F-NTA enables absolute counting of HCC-specific exosomes in the clinical samples with preserved biological immunoreactivity. The performance of F-NTA was clinically validated in serum from patients ± cirrhosis and with confirmed HCC. F-NTA quantification data show selective enrichment of AFP and GPC3 positive EVs in HCC serum compared to malignancy-free cirrhosis (AUC values for GPC3, AFP, and GPC3/AFP were found 0.79, 0.71, and 0.72 respectively). The MRI-confirmed patient cohort indicated that there was a positive correlation between total tumor size and GPC3-positive exosome concentration (r:0.78 and p<0.001).

Conclusion: We developed an immunocapture assay that can be used for simultaneous isolation and quantification of HCC-derived exosomes from a small serum volume with high accuracy.

Keywords: AFP; AUC; EVs; GPC3; HCC; MRI; alpha-fetoprotein; area under the curve; extracellular vesicles; glypican 3; hepatocellular carcinoma; magnetic resonance imaging.

Grants and funding

I01 BX004516/BX/BLRD VA/United States