Experimental Validation of Novel Glypican 3 Exosomes for the Detection of Hepatocellular Carcinoma in Liver Cirrhosis

Abstract

Background 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 malignancyfree 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.

Impaired Autophagy Response in Hepatocellular Carcinomas Enriches Glypican-3 in Exosomes, Not in the Microvesicles

“…Our laboratory recently showed that exosomal GPC3 is closely related to the impairment of autophagy by using ELISA analysis of HCC samples. 33 However, using the immuno-magnetic affinity assay, we show that GPC3 is restricted to the exosomal EV fraction both in the hepatoma cell lines and in the HCC samples. This suggests that GPC3 plays a major role in the biogenesis of multivesicular bodies and exosome release.…”

“…We previously showed that GPC3 expressing exosomes can be detected in the serum of patients with HCC cirrhosis. 33 Solid malignancies, including HCC, excrete both exosomes and microvesicles but which type of particle is selected by HCC for releasing GPC3 is not elucidated. Using the immuno-magnetic affinity assay, we examined both EV fractions in HCC cell culture supernatants.…”

Diagnostic accuracy and prognostic significance of Glypican-3 in hepatocellular carcinoma: A systematic review and meta-analysis

“…GPC-3, Glypican-3. showed that the expression of p62 and GPC-3 was significantly increased in HCC tissues compared with adjacent cirrhotic liver, and GPC-3-positive exosomes can be used for HCC detection and prediction of treatment outcomes (78). Moreover, data from the study by Montalbano reveal new aspects of the role of GPC-3 in early hepatocyte transformation.…”

GPC3 Electrochemical Aptasensor Based on Pd-Pt NPs@ RGO-H Nanozyme to Catalyze Silver Deposition

“…Glypican-3 (GPC3), a cell membrane-anchored oncofetal proteoglycan, is a novel marker of primary hepatocellular carcinoma (HCC) that outperforms the clinically commonly used alpha-fetoprotein (AFP) and performs satisfactorily in the early clinical screening of HCC [1]. However, the current methods for detecting GPC3 are mainly enzyme-linked immunoassay (ELISA), bioluminescence enzyme immunoassay (BLEIA), etc., but these methods have the disadvantages of low sensitivity, poor stability, and susceptibility to multiple factors [2,3]. Therefore, there is an urgent need for a new method of GPC3 detection that is fast and sensitive and easy to operate.…”