Journal of Hepatocellular Carcinoma (Nov 2023)
A Single-Step Immunocapture Assay to Quantify HCC Exosomes Using the Highly Sensitive Fluorescence Nanoparticle-Tracking Analysis
Abstract
Ali Riza Koksal,1 Nergiz Ekmen,2 Yucel Aydin,2 Kelley Nunez,3 Tyler Sandow,4 Molly Delk,2 Martin Moehlen,2 Paul Thevenot,3 Ari Cohen,3,5 Srikanta Dash1,2,6 1Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, New Orleans, LA, USA; 2Department of Gastroenterology and Hepatology, Tulane University Health Sciences Center, New Orleans, LA, USA; 3Department of Gastroenterology and Hepatology, Institute of Translational Research, Ochsner Health, New Orleans, LA, USA; 4Department of Radiology, Institute of Translational Research, Ochsner Health, New Orleans, LA, USA; 5Multi-Organ Transplant Institute, Ochsner Health, New Orleans, LA, USA; 6Southeast Louisiana Veterans Health Care System, New Orleans, LA, USACorrespondence: Srikanta Dash, Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA, 70112, USA, Tel +1 504-988-2519, Fax +1 504-988-7389, Email [email protected]: 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: hepatocellular carcinoma, HCC, extracellular vesicles, EVs, glypican 3, GPC3, alpha-fetoprotein, AFP, magnetic resonance imaging, MRI, area under the curve, AUC
