Detection of circulating tumor cells in hepatocellular carcinoma using antibodies against asialoglycoprotein receptor, carbamoyl phosphate synthetase 1 and pan-cytokeratin.

Linlin Zhao,Huiying Liu,Bin Sun,Naijian Ge,Xiaofeng Zhang,Zhengfeng Yin,Xin-Yuan Guan,Jun Li,Yefa Yang,Yu Zhang,Lei Chen,Mengchao Wu,Haihua Qian

doi:10.1371/journal.pone.0096185

Abstract

BackgroundAsialoglycoprotein receptor (ASGPR)-ligand-based separation combined with identification with Hep Par 1 or pan-cytokeratin (P-CK) antibody have been demonstrated to detect circulating tumor cells (CTCs) in hepatocellular carcinoma (HCC). The aim of this study was to develop an improved enrichment and identification system that allows the detection of all types of HCC CTCs.MethodsThe specificity of the prepared anti-ASGPR monoclonal antibody was characterized. HCC cells were bound by ASGPR antibody and subsequently magnetically isolated by second antibody-coated magnetic beads. Isolated HCC cells were identified by immunofluorescence staining using a combination of anti-P-CK and anti-carbamoyl phosphate synthetase 1 (CPS1) antibodies. Blood samples spiked with HepG2 cells were used to determine recovery and sensitivity. CTCs were detected in blood samples from HCC patients and other patients.ResultsASGPR was exclusively expressed in human hepatoma cell line, normal hepatocytes and HCC cells in tissue specimens detected by the ASGPR antibody staining. More HCC cells could be identified by the antibody cocktail for CPS1 and P-CK compared with a single antibody. The current approach obtained a higher recovery rate of HepG2 cells and more CTC detection from HCC patients than the previous method. Using the current method CTCs were detected in 89% of HCC patients and no CTCs were found in the other test subjects.ConclusionsOur anti-ASGPR antibody could be used for specific and efficient HCC CTC enrichment, and anti-P-CK combined with anti-CPS1 antibodies is superior to identification with one antibody alone in the sensitivity for HCC CTC detection.

Highlights

Circulating tumor cells (CTCs) are cancer cells shed from either the primary tumor or its metastases that circulate in the peripheral blood
Asialoglycoprotein receptor (ASGPR) was Exclusively Expressed in Human Hepatoma Cell Line, Normal Hepatocytes and hepatocellular carcinoma (HCC) Cells in Tissue Specimens Detected by the ASGPR Antibody Staining
The results showed that all the test cell lines derived from extrahepatic origin including MCF-7, A498, SW480, and NIH-3T3 didn’t bind the antibody (Fig. 1A), but on the contrary, the expression level of ASGPR can be measured in the human hepatoma cell line HepG2 (Fig. 1A), and the positive expression rate was (95.162.6)%

Summary

Introduction

Circulating tumor cells (CTCs) are cancer cells shed from either the primary tumor or its metastases that circulate in the peripheral blood. We have previously developed a unique magnetic HCC CTC separation system mediated by the interaction of the asialoglycoprotein receptor (ASGPR) with its ligand [21]. The ligand-receptor binding assay has its own disadvantages, which will limit its transformation of clinical practice in HCC CTC detection. Since an antibody-antigen binding assay is a better alternative, we prepared a monoclonal antibody specific for ASGPR, modified the magnetic HCC CTC separation method and detection approach, in which HCC CTCs were captured by using anti-ASGPR antibody. Asialoglycoprotein receptor (ASGPR)-ligand-based separation combined with identification with Hep Par 1 or pan-cytokeratin (P-CK) antibody have been demonstrated to detect circulating tumor cells (CTCs) in hepatocellular carcinoma (HCC).

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