title: Identification of biomarkers for hepatocellular carcinoma by semiquantitative immunocytochemistry.
abstract: OBJECTIVE
  To investigate the expression of key biomarkers in hepatoma cell lines, tumor cells from patients' blood samples, and tumor tissues.
  
  
  METHODS
  We performed the biomarker tests in two steps. First, cells plated on coverslips were used to assess biomarkers, and fluorescence intensities were calculated using the NIH Image J software. The measured values were analyzed using the SPSS 19.0 software to make comparisons among eight cell lines. Second, eighty-four individual samples were used to assess the biomarkers' expression. Negative enrichment of the blood samples was performed, and karyocytes were isolated and dropped onto pre-treated glass slides for further analysis by immunofluorescence staining. Fluorescence intensities were compared among hepatocellular carcinoma (HCC) patients, chronic HBV-infected patients, and healthy controls following methods similar to those used for cell lines. The relationships between the expression of biomarkers and clinical pathological parameters were analyzed by Spearman rank correlation tests. In addition, we studied the distinct biomarkers' expression with three-dimensional laser confocal microscopy reconstructions, and Kaplan-Meier survival analysis was performed to understand the clinical significance of these biomarkers.
  
  
  RESULTS
  Microscopic examination and fluorescence intensity calculations indicated that cytokeratin 8/18/19 (CK) expression was significantly higher in six of the seven HCC cell lines examined than in the control cells, and the expression levels of asialoglycoprotein receptor (ASGPR) and glypican-3 (GPC3) were higher in all seven HCC cell lines than in the control. Cells obtained from HCC patients' blood samples also displayed significantly higher expression levels of ASGPR, GPC3, and CK than cells from chronic HBV-infected patients or healthy controls; these proteins may be valuable surface biomarkers for identifying HCC circulating tumor cells isolated and enriched from the blood samples. The stem cell-like and epithelial-mesenchymal transition-related biomarkers could be detected on the karyocyte slides. ASGPR and GPC3 were expressed at high levels, and thus three-dimensional reconstructions were used to observe their expression in detail. This analysis indicated that GPC3 was localized in the cytoplasm and membrane, but that ASGPR had a polar localization. Survival analyses showed that expression of GPC3 and ASGPR is associated with a patient's overall survival (OS).
  
  
  CONCLUSIONS
  ASGPR, GPC3, and CK may be valuable HCC biomarkers for CTC detection; the expression of ASGPR and GPC3 might be helpful for understanding patients' OS.
authors.affiliation: Hong Mu, Fang Liu, Yu-Lin Sun, Xiao-Hang Zhao, State Key Laboratory of Molecular Oncology, Cancer Institute Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Mu
authors.affiliation: Hong Mu, Fang Liu, Yu-Lin Sun, Xiao-Hang Zhao, State Key Laboratory of Molecular Oncology, Cancer Institute Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China.
authors.forename: Kai-Xuan
authors.initials: KX
authors.lastname: Lin
authors.affiliation: Hong Mu, Fang Liu, Yu-Lin Sun, Xiao-Hang Zhao, State Key Laboratory of Molecular Oncology, Cancer Institute Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Zhao
authors.affiliation: Hong Mu, Fang Liu, Yu-Lin Sun, Xiao-Hang Zhao, State Key Laboratory of Molecular Oncology, Cancer Institute Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China.
authors.forename: Shu
authors.initials: S
authors.lastname: Xing
authors.affiliation: Hong Mu, Fang Liu, Yu-Lin Sun, Xiao-Hang Zhao, State Key Laboratory of Molecular Oncology, Cancer Institute Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China.
authors.forename: Cong
authors.initials: C
authors.lastname: Li
authors.affiliation: Hong Mu, Fang Liu, Yu-Lin Sun, Xiao-Hang Zhao, State Key Laboratory of Molecular Oncology, Cancer Institute Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China.
authors.forename: Fang
authors.initials: F
authors.lastname: Liu
authors.affiliation: Hong Mu, Fang Liu, Yu-Lin Sun, Xiao-Hang Zhao, State Key Laboratory of Molecular Oncology, Cancer Institute Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China.
authors.forename: Hai-Zhen
authors.initials: HZ
authors.lastname: Lu
authors.affiliation: Hong Mu, Fang Liu, Yu-Lin Sun, Xiao-Hang Zhao, State Key Laboratory of Molecular Oncology, Cancer Institute Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China.
authors.forename: Ze
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Hong Mu, Fang Liu, Yu-Lin Sun, Xiao-Hang Zhao, State Key Laboratory of Molecular Oncology, Cancer Institute Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China.
authors.forename: Yu-Lin
authors.initials: YL
authors.lastname: Sun
authors.affiliation: Hong Mu, Fang Liu, Yu-Lin Sun, Xiao-Hang Zhao, State Key Laboratory of Molecular Oncology, Cancer Institute Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China.
authors.forename: Xi-Yun
authors.initials: XY
authors.lastname: Yan
authors.affiliation: Hong Mu, Fang Liu, Yu-Lin Sun, Xiao-Hang Zhao, State Key Laboratory of Molecular Oncology, Cancer Institute Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China.
authors.forename: Jian-Qiang
authors.initials: JQ
authors.lastname: Cai
authors.affiliation: Hong Mu, Fang Liu, Yu-Lin Sun, Xiao-Hang Zhao, State Key Laboratory of Molecular Oncology, Cancer Institute Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China.
authors.forename: Xiao-Hang
authors.initials: XH
authors.lastname: Zhao
mesh_terms: D000328:Adult; D000368:Aged; D037263:Asialoglycoprotein Receptor / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000175:diagnosis* / Q000378:metabolism / Q000401:mortality; D045744:Cell Line, Tumor; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D053673:Glypicans / Q000378:metabolism; D006515:Hepatitis B virus; D019694:Hepatitis B, Chronic / Q000150:complications / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D053208:Kaplan-Meier Estimate; D053538:Keratin-18 / Q000378:metabolism; D053539:Keratin-19 / Q000378:metabolism; D053533:Keratin-8 / Q000378:metabolism; D008113:Liver Neoplasms / Q000175:diagnosis* / Q000378:metabolism / Q000401:mortality; D008297:Male; D008875:Middle Aged; D009362:Neoplasm Metastasis; D009364:Neoplasm Recurrence, Local; D009360:Neoplastic Cells, Circulating / Q000378:metabolism
chemical_list: D037263:Asialoglycoprotein Receptor; D014408:Biomarkers, Tumor; D053673:Glypicans; D053538:Keratin-18; D053539:Keratin-19; D053533:Keratin-8
keywords: Biomarker; Hepatocellular carcinoma; Immunocytochemistry; Semiquantitative analysis; Three-dimensional reconstruction