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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 sdash@tulane.eduIntroduction: 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

Ali Riza Koksal,1,2 Paul Thevenot,3 Yucel Aydin,1 Kelley Nunez,3 Tyler Sandow,4 Kyle Widmer,5 Leela Nayak,5 John Scott,1 Molly Delk,2 Martin W Moehlen,2 Ari J Cohen,3,6 Srikanta Dash1,2,5 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, Multi-Organ Transplant Institute, Ochsner Health, New Orleans, LA, USA; 5Southeast Louisiana Veterans Health Care System, New Orleans, LA, USA; 6Department of General Surgery, Multi-Organ Transplant Institute, Ochsner Health, 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 sdash@tulane.eduBackground and Aim: HCC development in liver cirrhosis is associated with impaired autophagy leading to increased production of extracellular vesicles (EVs) including exosomes and microvesicles. The goal of the study is to determine which of these particles is primarily involved in releasing of HCC-specific biomarker glypican-3 (GPC3) when autophagy is impaired.Methods: Streptavidin-coated magnetic beads were coupled with either biotinylated CD63 or Annexin A1 antibodies. Coupled beads were incubated with EVs isolated from either HCC culture or serum. EVs captured by immuno-magnetic beads were then stained with FITC or PE fluorescent-conjugated antibodies targeting exosomes (CD81), and microvesicles (ARF6). The percentage of GPC3 enrichment in the microvesicles and exosomes was quantified by flow cytometry. The impact of autophagy modulation on GPC3 enrichment in exosomes and microvesicles was assessed by treating cells with Torin 1 and Bafilomycin A1. For clinical validation, GPC3 content was quantified in microvesicles, and exosomes were isolated from the serum of patients with a recent HCC diagnosis.Results: The immune-magnetic bead assay distinguishes membrane-derived microvesicles from endosome-derived exosomes. The GPC3 expression was only seen in the CD63 beads group but not in the Annexin A1 beads group, confirming that in HCC, GPC3 is preferentially released through exosomes. Furthermore, we found that autophagy induction by Torin1 decreased GPC3-positive exosome secretion and decreased microvesicle release. Conversely, autophagy inhibition by Bafilomycin A1 increased the secretion of GPC3-positive exosomes. Serum analysis showed CD81+ve EVs were detected in exosomes and ARF6+ve vesicles were detected in microvesicles, suggesting that immunoaffinity assay is specific. The exosomal GPC3 enrichment was confirmed in isolated EVs from the serum of patients with HCC. The frequency of GPC3-positive exosomes was higher in patients with HCC (12.4%) compared to exosomes isolated from non-cirrhotic and healthy controls (3.7% and 1.3% respectively, p< 0.001).Conclusion: Our results show that GPC3 is enriched in the endolysosomal compartment and released in exosome fractions when autophagy is impaired.Keywords: hepatocellular carcinoma, glypican 3, exosome, biomarker, magnetic beads

Yucel Aydin,1 Ali Riza Koksal,1,2 Paul Thevenot,3 Srinivas Chava,1 Zahra Heidari,4 Dong Lin,1 Tyler Sandow,5 Krzysztof Moroz,1 Mansour A Parsi,2 John Scott,1 Ari Cohen,3,6 Srikanta Dash1,7 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; 3Institute of Translational Research, Ochsner Health, New Orleans, LA, USA; 4Chemical and Biomedical Engineering, Tulane University, New Orleans, LA, USA; 5Department of Radiology, Institute of Translational Research, Ochsner Health, New Orleans, LA, USA; 6Multi-Organ Transplant Institute, Ochsner Health, New Orleans, LA, USA; 7Southeast Louisiana Veterans Health Care System, New Orleans, LA, USACorrespondence: Srikanta Dash Email sdash@tulane.eduBackground and Aims: Hepatocellular carcinoma (HCC) developing in the context of preexisting cirrhosis is characterized by impaired autophagy that results in increased exosome release. This study was conducted to determine whether circulating exosomes expressing glypican 3 (GPC3) could be utilized as a biomarker for HCC detection and treatment response in patients with cirrhosis.Methods: Immunohistochemistry was performed to assess p62 and GPC3 expression in the lesion and adjacent tissue from cirrhosis with HCC. GPC3-enriched exosomes were captured by an enzyme-linked immunosorbent assay (ELISA). The diagnostic specificity of serum exosome-derived GPC3 (eGPC3) was determined using samples obtained from malignancy-free controls, malignancy-free cirrhotics, cirrhotics with confirmed HCC, and patients with a non-HCC malignancy. The performance of eGPC3 was validated using serum samples of HCC patients received chemotherapy.Results: We found that the expression of p62 and GPC3 was significantly increased in HCC tissues compared to adjacent cirrhotic liver. Impaired autophagy and exosome shedding were confirmed in HCC cell lines. Mass spectroscopic analysis revealed that GPC3 was enriched in exosomes isolated from HCC cell lines. An affinity ELISA assay was developed that specifically captures GPC3 positive exosomes in the serum. Total exosome concentration and eGPC3 were significantly elevated in cirrhotic patients with HCC as compared to the reference control groups. Furthermore, decreases in post-treatment exosome concentration and eGPC3 levels were more closely correlated with response to locoregional chemotherapy compared to change in serum AFP in HCC patients awaiting liver transplantation.Conclusion: We developed an affinity exosome capture assay to detect GPC3 enriched exosomes. Our preliminary assessment shows that GPC3 positive exosomes can be used for HCC detection and prediction of treatment outcomes.Keywords: hepatocellular carcinoma, autophagy, exosome, glypican 3, surveillance

Aydin Y, Koksal AR, Thevenot P, et al. J Hepatocell Carcinoma. 2020;15:1771–1786. The authors have advised due to an error that occurred inadvertently at the time of figure assembly, Figure 3 on page 1587 is incorrect. The high magnification images of Huh-7.5 and HLE should be interchanged. The correct Figure 3 is shown below. Figure 3 Characterization of exosomes released from an autophagy-deficient and an autophagy-competent HCC cell line. (A) Cryogenic transmission electron microscopy (cryoTEM) images of exosomes purified from culture supernatants of autophagy-deficient (Huh-7.5) and autophagy-competent (HLE) HCC lines. Left panel (low magnification). Right panel (high magnification). (B) Top panel: Representative image of exosome Brownian motion in liquid phase using Nanoparticle tracking analysis (NTA). Bottom panel: Size distribution profile of exosomes with mean and mode of the population diameter ± standard error. (C) Exosome concentration in the culture supernatant quantified by NTA. The results are expressed as the mean ± standard deviation and analyzed by Student’s t-test. *P < 0.05. (D) Representative Western blot for TSG101 and CD9 in cell lysates and exosomes isolated from autophagy-deficient Huh-7.5 and autophagy-competent HLE cell cultures. All experiments were performed in triplicate. The authors apologize for the error and advise it does not affect the results and conclusion of the paper.