| Background and ObjectiveCirculating tumor cells (CTCs) in patients with hepatocellular carcinoma (HCC) are regarded to be the root of metastases and recurrences of HCC. Therefore, the detection of CTCs in HCC patients undoubtedly has considerable clinical significance in predicting recurrences and metastases. Current strategies for detecting CTCs in HCC patients are limited to complex analytic approaches based on reverse-transcription polymerase chain reaction (RT-PCR), which yield inevitable false positive and negative results. Moreover, the RT-PCR-based assays cannot accurately quantify CTCs and no morphologic evaluation of the cells can be obtained. In summary, it is imperative to establish a sensitive and specific method to isolate and detect CTCs in HCC patients. Immunomagnetic bead separation based on antibodies for tumor cell surface antigens is currently the standard method employed to detect CTCs in the clinical setting. Given that there are currently no antibodies specific for HCC cell surface antigens, few reports have been published on the separation of CTCs in HCC patients by this method. Epithelial cell adhesion molecule (EpCAM) is an epithelial cell-specific adhesion molecule that is widely expressed on the surface of epithelial cells and epithelial-derived tumor cells. The CellSearch system, based on EpCAM antibody-coated magnetic beads, has been approved by the US Food and Drug Administration for detection of CTCs in breast cancer, colon cancer and prostate cancer. Although HCC cells are epithelial cells, only about 35% of HCC cases express EpCAM. Therefore, the CellSearch system is not appropriate for detection of CTCs in HCC patients. In summary, it is imperative to establish a sensitive and specific method to detect CTCs in HCC patients.The asialoglycoprotein receptor (ASGPR) is a transmembrane protein exclusively expressed on the surface of hepatocytes, which can bind and internalize molecules with terminal galactose and N-acetylgalactosamine residues. By taking advantage of this characteristic, liver targeting systems for drugs and genes have been developed using glycosylated macromolecules as vehicles that can be selectively recognized by ASGPR. Here we describe the development and validation of a sensitive and specific system to magnetically separate CTCs in HCC patients, mediated by the interaction of the ASGPR with its ligand. In the system, HCC cells were bound by biotinylated asialofetuin, an ASGPR ligand, and subsequently labeled by anti-biotin antibody-coated magnetic beads, followed by magnetic separation. The separated HCC cells were then identified by immunofluorescence staining using the hepatocyte-specific antibody Hep Par 1. Given that normal hepatocytes do not circulate, unless they become tumorous, any of the cells detected by the system are circulating HCC cells. This system solves the inherent problems of previously reported methods and has high sensitivity and specificity. To evaluate the feasibility of its using in the clinic, we have applied this system to patients with HCC, patients with benign liver diseases and healthy volunteers.Methods1. Biotinylated asialofetuin was made by biotinylating asialofetuin with sulfo-NHS-LC-biotin, and then purified by using centrifugal ultrafiltration.2. Flow cytometry was used to validate the binding activity of biotinylated asialofetuin to HCC cells, and to determine its optimal working concentration.3. The specificity of binding of biotinylated asialofetuin to HCC cells was analyzed by flow cytometry.4. For magnetic separation, the hepatoma carcinoma cells were bound by biotinylated asialofetuin and subsequently labeled by anti-biotin antibody-coated magnetic mircobeads, followed by magnetic separation.5. The separated HCC cells were identified by immunofluorescence staining using the hepatocyte-specific antibody Hep Par 1.6. The recovery, specificity and sensitivity of the HCC CTCs separation and detection system were determined by the Hep3B cell spiking experiments.7. The recovery of the separation method based on ASGPR and that of the separation methed based on EpCAM antibody-coated magnetic microbeads were compared by Hep3B cell spiking experiments.8. The established separation and detection system for HCC CTCs was applied to examine the prevalence of CTCs in blood samples from 235 patients with HCC,20 healthy volunteers,37 patients with benign liver diseases (including 16 patients with cirrhosis,4 patients with chronic hepatitis B,6 patients with acute hepatitis A,8 patients with hepatic hemangioma and 3 patients with liver cysts) and 19 patients with non-HCC cancers (3 testicular,4 thyroid,4 colon,3 kidney and 5 metastatic breast cancer).9. The system was applied to detect CTCs in 11 patients with benign hepatic space-occupying lesions (8 patients with hepatic hemangioma and 3 patients with liver cysts) 1 day,4 days,7 days,10 days and 14 days after surgery.10. The TUNEL assay was used to identify apoptotic CTCs in HCC patients.11. Immunofluorescence staining with CD 133 antibody was employed to identify cancer stem cells in CTCs from HCC patients.12. Spearman rank correlation analysis and Fisher's exact test were used to analyze the correlation between the positivity rate of CTCs and the clinical parameters. Spearman rank correlation analysis and Mann-Whitney test were used to analyze the correlation between the number of CTCs and the clinical parameters.Results1. Flow cytometry results showed the good binding activity of biotinylated asialofetuin to HCC cells, and its optimal working concentration was 0.4 mg/mL.2. Flow cytometry results suggested high specificity of the binding of biotinylated asialofetuin to HCC cells.3. The cell spiking experiments showed that when there were 10,30,90,270 and 810 Hep3B cells spiked into five mL of peripheral blood from healthy volunteers, the average recovery was≥61% at each of the spiking levels, and in samples spiked with 10 cells, no fewer than 5 cells were detected in all five samples.4. In all samples spiked with 100 MCF-7 breast cancer cells or 100 A498 kedney cancer cells, none had cytokeratin-positive cells detected.5. The cell spiking experiments showed that the recovery of the separation method based on ASGPR was higher than that of the separation methed based on EpCAM antibody-coated magnetic microbeads (P<0.05).6. No CTCs were detected in any blood samples from 20 healthy volunteers,37 patients with benign liver diseases or 19 patients with non-HCC cancers.7. Among 11 patients with benign hepatic space-occupying lesions (8 patients with hepatic hemangioma and 3 patients with liver cysts), none had CTCs detected before surgery, and all had Hep Par 1-positive cells detected on day 1,4,7, and 10 after surgery. The number of Hep Par 1-positive cells detected decreased gradually from day 1 to day 10 after surgery, and no Hep Par 1-positive cells were detected 14 days after surgery in each of the 11 patients.8. CTCs were detected in the blood samples from 195 of 235 (83%) patients with HCC. The number of CTCs detected ranged from 0 to 153 per 5 mL peripheral blood, with an average of 21±25 (mean±SD).9. CD133-positive CTCs were detected in some HCC patients.10. Some of the CTCs in HCC patients were apoptotic.11. Statistic analyses showed that both the positivity rate and the number of CTCs were significantly correlated with tumor size, portal vein tumor thrombus, and the disease extent as classified by the TNM classification and the Milan criteria, but neither the positivity rate nor the number of CTCs were correlated with age, sex, etiology, Child-Pugh class or serum AFP level.ConclusionsWe have developed a new tool capable of highly sensitive and specific separation and detection of CTCs in HCC patients. It is likely clinically useful in diagnosis and monitoring of HCC. Both the positivity rate and the number of CTCs are significantly correlated with the disease extent of HCC. Liver resection can induce release of liver cells into the peripheral blood circulation, and the non-tumorous cells will disappear within about 2 weeks. Some of the CTCs in HCC patients were apoptotic, and CTCs with liver cancer stem cell phynotype were detected in some HCC patients.
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