Detection of circulating tumor cells in hepatocellular carcinoma using antibodies against asialoglycoprotein receptor, carbamoyl phosphate synthetase 1 and pan-cytokeratin.
BACKGROUND
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). The aim of this study was to develop an improved enrichment and identification system that allows the detection of all types of HCC CTCs.


METHODS
The 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.


RESULTS
ASGPR 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.


CONCLUSIONS
Our 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.
Molecular Oncology Laboratory, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China.
Jun
J
Li
Molecular Oncology Laboratory, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China.
Lei
L
Chen
Molecular Oncology Laboratory, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China.
Xiaofeng
X
Zhang
Molecular Oncology Laboratory, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China.
Yu
Y
Zhang
Molecular Oncology Laboratory, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China.
Huiying
H
Liu
Molecular Oncology Laboratory, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China.
Bin
B
Sun
Molecular Oncology Laboratory, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China.
Linlin
L
Zhao
Department of Interventional Radiology, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China.
Naijian
N
Ge
Molecular Oncology Laboratory, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China.
Haihua
H
Qian
Department of Interventional Radiology, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China.
Yefa
Y
Yang
Molecular Oncology Laboratory, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China; Department of Interventional Radiology, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China.
Mengchao
M
Wu
Molecular Oncology Laboratory, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China.
Zhengfeng
Z
Yin
D000818:Animals; D000906:Antibodies; D037263:Asialoglycoprotein Receptor / Q000276:immunology / Q000378:metabolism*; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000276:immunology / Q000378:metabolism*; D006528:Carcinoma, Hepatocellular / Q000097:blood / Q000473:pathology*; D045744:Cell Line, Tumor; D005260:Female; D006801:Humans; D007633:Keratins / Q000276:immunology / Q000378:metabolism*; D008113:Liver Neoplasms / Q000097:blood / Q000473:pathology*; D008807:Mice, Inbred BALB C; D009360:Neoplastic Cells, Circulating / Q000276:immunology / Q000473:pathology*
D000906:Antibodies; D037263:Asialoglycoprotein Receptor; D007633:Keratins; D002222:Carbamoyl-Phosphate Synthase (Ammonia)