Basic Study
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Mar 14, 2015; 21(10): 2918-2925
Published online Mar 14, 2015. doi: 10.3748/wjg.v21.i10.2918
Improved method increases sensitivity for circulating hepatocellular carcinoma cells
Hui-Ying Liu, Hai-Hua Qian, Xiao-Feng Zhang, Jun Li, Xia Yang, Bin Sun, Jun-Yong Ma, Lei Chen, Zheng-Feng Yin
Hui-Ying Liu, Hai-Hua Qian, Xiao-Feng Zhang, Jun Li, Xia Yang, Bin Sun, Jun-Yong Ma, Lei Chen, Zheng-Feng Yin, Molecular Oncology Laboratory, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China
Author contributions: Liu HY and Qian HH contributed equally to this work; Yin ZF designed the study; Liu HY, Qian HH, Zhang XF, Li J and Yang X collected and analyzed the data; Liu HY, Qian HH, Zhang XF, Li J, Yang X, Sun B, Ma JY, Chen L and Yin ZF interpreted the results of the study; Liu HY and Yin ZF wrote the manuscript.
Supported by Grants from the China National Key Projects for Infectious Disease, No. 2012ZX10002012-10; and the National Nature Science Foundation of China, No. 81172207, No. 81272669 and No. 81301830.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Zheng-Feng Yin, MD, PhD, Molecular Oncology Laboratory, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, 225 Changhai Road, Shanghai 200438, China. yinzfk@aliyun.com
Telephone: +86-21-81875354 Fax: +86-21-81875354
Received: August 4, 2014
Peer-review started: August 4, 2014
First decision: August 15, 2014
Revised: September 10, 2014
Accepted: November 30, 2014
Article in press: December 1, 2014
Published online: March 14, 2015
Abstract

AIM: To improve an asialoglycoprotein receptor (ASGPR)-based enrichment method for detection of circulating tumor cells (CTCs) of hepatocellular carcinoma (HCC).

METHODS: Peripheral blood samples were collected from healthy subjects, patients with HCC or various other cancers, and patients with hepatic lesions or hepatitis. CTCs were enriched from whole blood by extracting CD45-expressing leukocytes with monoclonal antibody coated-beads following density gradient centrifugation. The remaining cells were cytocentrifuged on polylysine-coated slides. Isolated cells were treated by triple immunofluorescence staining with CD45 antibody and a combination of antibodies against ASGPR and carbamoyl phosphate synthetase 1 (CPS1), used as liver-specific markers, and costained with DAPI. The cell slide was imaged and stained tumor cells that met preset criteria were counted. Recovery, sensitivity and specificity of the detection methods were determined and compared by spiking experiments with various types of cultured human tumor cell lines. Expression of ASGPR and CPS1 in cultured tumor cells and tumor tissue specimens was analyzed by flow cytometry and triple immunofluorescence staining, respectively.

RESULTS: CD45 depletion of leukocytes resulted in a significantly greater recovery of multiple amounts of spiked HCC cells than the ASGPR+ selection (Ps < 0.05). The expression rates of either ASGPR or CPS1 were different in various liver cancer cell lines, ranging between 18% and 99% for ASGPR and between 9% and 98% for CPS1. In both human HCC tissues and liver cancer cell lines, there were a few HCC cells that did not stain positive for ASGPR or CPS1. The mixture of monoclonal antibodies against ASGPR and CPS1 identified more HCC cells than either antibody alone. However, these antibodies did not detect any tumor cells in blood samples spiked with the human breast cancer cell line MCF-7 and the human renal cancer cell line A498. ASGPR+ or/and CPS1+ CTCs were detected in 29/32 (91%) patients with HCC, but not in patients with any other kind of cancer or any of the other test subjects. Furthermore, the improved method detected a higher CTC count in all patients examined than did the previous method (P = 0.001), and consistently achieved 12%-21% higher sensitivity of CTC detection in all seven HCC patients with more than 40 CTCs.

CONCLUSION: Negative depletion enrichment combined with identification using a mixture of antibodies against ASGPR and CPS1 improves sensitivity and specificity for detecting circulating HCC cells.

Keywords: Asialoglycoprotein receptor, Carbamoyl phosphate synthetase 1, Circulating tumor cells, Hepatocellular carcinoma, Negative depletion

Core tip: We previously described asialoglycoprotein receptor (ASGPR)-based positive selection for the enrichment and further detection of circulating tumor cells (CTCs) of hepatocellular carcinoma (HCC). However, expression of ASGPR is heterogeneous in human HCC, and ASGPR- cells are missed by this method. In this study, we describe an improved method using depletion enrichment with identification using antibodies against ASGPR and carbamoyl phosphate synthetase 1. The improved method significantly improved sensitivity for CTC enrichment, and also provided high specificity for CTC detection in patients with HCC, thereby minimizing false negative/positive results.