| Background&ObjectiveHepatocellular carcinoma (HCC) is one of the world’s most common cancer, withhigh degree of malignancy. There are about13million people died of liver cancer andrelated diseases each year in China. Despite the current use of hepatectomy to improvethe long-term survival, there is still a high incidence of recurrence because ofpostsurgical recurrence. Many studies have shown that tumor cells have been shed intothe blood in part of the HCC patients before curative resection of liver cancer, which is anecessary prerequisite that lead to blood metastasis. Tumor cells that shed from theprimary tumor and circulate through the bloodstream are called circulating tumor cells(CTCs) that are the root causes of recurrence and metastasis. As the link between primarytumor and metastases, and the window between tumor biology and metastasis, CTCs willnot only be a irreplaceable tumor biomarker for the guiding individualized treatment, butalso is an important point for the mechanisms study of tumor recurrence and metastasisprocesses. Given epithelial-mesenchymal transition (EMT) is a key factor in promotingmigration by the induction of CTC, CTC molecular diagnostics, targeted research andapplication of treatment are received more and more attention. It could be metastatic andprognostic molecular markers of a variety of malignant tumors.We have previously developed a system to magnetically separate HCC CTCs,mediated by the interaction of the asialoglycoprotein receptor (ASGPR) with its ligand. The HCC CTCs were then identified using the hepatocyte-specific antibody Hep Par1alone by immunofluorescence staining. However, the ligand-receptor binding assayhas its own disadvantages. We prepared a monoclonal antibody specific for ASGPR,modified the magnetic HCC CTCs separation method and detection approach, in whichHCC CTCs were captured by using anti-ASGPR antibody. The HCC CTCs were thenidentified using a combination of anti-carbamoyl phosphate synthetase1(CPS1) and anti-P-CK antibodies, which had a high detection rate. In the first part of this study, wehave used a combination of anti-carbamoyl phosphate synthetase1(CPS1) andanti-ASGPR antibodies instead of combination of anti-carbamoyl phosphate synthetase1(CPS1) and anti-P-CK antibodies, because we have found CKs potential false positivesand heterogeneity CPS1expression in HCC, reducing the separation efficiency of theidentification. For positive sorting, ASGPR was expressed on intra-hepatic cell lines atvarying levels, reducing recovery efficiency. We used MACS-negative sorting methodinstead of positive sorting, thus avoiding of tumor heterogeneity on enrichment efficiency.The recovery, specificity and sensitivity of the current method were determined by theHepG2cell spiking experiments. The recovery of the separation method based on theimproved method and the original method were compared by HepG2cell spikingexperiments. And it was applied to the detection of CTCs in HCC patients, comparingwith the original method. The second part of this work aims to establish a multi-colorimmunofluorescent detecion model of CTCs, utilizing anti-CPS1and anti-ASGPR as anidentifying marker, and simultaneously detecting the expression of EMT markers inCTCs. And we continue to explore the value of the separation and detection system topredict the risk of liver cancer recurrence after radical, as a guide to developindividualized treatment plan.Methods1. CPS1and ASGPR expression in hepatoma cell lines were detected by flow cytometricanalysis.2. CPS1and ASGPR expression in hepatoma tissue by triple-fluorescentimmunohistochemistry.3. Mononuclear cells and tumor cells were enriched from the whole blood samples bydensity gradient Ficoll-Paque PLUS.4. For magnetic separation, the leukocyte cells were bound by CD45antibody-coatedmagnetic mircobeads, followed by magnetic separation.5. The separated HCC cells were identified by immunofluorescence staining using the hepatocyte-specific antibody CPS1and ASGPR.6. The recovery, specificity and sensitivity of the current method were determined by theHepG2cell spiking experiments.7. The recovery of the separation method based on the improved method and the originalmethod were compared by HepG2cell spiking experiments.8. The current method was applied to detect CTCs in27HCC patients. The recovery ofthe current method and original method were compared by10HCC patients.9. Several HCC cell lines were used in cell-spiking experiment to establish amulti-immunofluorescence staining model of CTCs, utilizing CPS1and ASGPR as anidentifying marker, and simultaneously detecting the expression of Zeb1and Vimentin.10. Five milliliters of the whole blood for CTCs detection was drawn from all86patientsbefore the first operation. And they were triple stained by CPS1and ASGPR antibodyalong with Zeb1and Vimentin antibodies. Correlation analysis of the expression of Zeb1and Vimentin in HCC.11. Serial tissue sections of86HCC patients were examined by immunohistochemistryand immunofluorescent detecion of Zeb1/Vimentin expression. The results obtained fromHCC tissues and CTCs were compared.12. Immunofluorescence staining was employed to identify Zeb1and Vimentinexpression in CTM from several HCC patients.13. Regular review and follow-up.Results1. Applicated ASGPR and CPS1antibodies to detect the positive rate of HCC cells, itwas higher than a single antibody.2. Applicated ASGPR and CPS1antibody to detect HCC tissues could avoid falsenegative of single antibody detection.3. The cell spiking experiments showed that the average recovery was≥80%at each ofthe spiking levels, and in samples spiked with10cells, no fewer than5cells weredetected in all five samples. In all samples spiked with100MCF-7breast cancer cells or 100A498kedney cancer cells, none had CPS1and ASGPR-positive cells detected.4. The cell spiking experiments showed that the recovery of the separation method basedon improved method with was higher than that of the original method (P<0.05).5. CTCs were detected in the blood samples from24of27(89%) patients with HCC. Thenumber of CTCs detected ranged from0to5per5mL peripheral blood, with an averageof33±26. No CTCs were detected in any blood samples from20healthy volunteers,40patients with benign liver diseases or17patients with non-HCC cancers.6. The new method could detected more number of CTCs compared to the originalmethod in10patients (P <0.01). And it increased16-26%sensitivity compared to theprevious method.7. Created CPS1+ASGPR/Zeb1/Vimentin multicolor immunofluorescence method werenot only able to identify CTCs but also were able to detect Zeb1/Vimentin phenotype. Itcan accurately detect Zeb1and Vimentin differential expression between different cells.8. CTCs were detected in the blood samples from75of86(87%) patients with HCC. Thenumber of CTCs detected ranged from3to96per5mL peripheral blood, with anaverage of33±17in75HCC patients. The expression of Zeb1and Vimentin diversifiedin CTCs from75HCC patients. Accordingly, they could be divided into four types asZeb1+/Vimentin+, Zeb1+/Vimentin-, Zeb1-/Vimentin+and Zeb1-/Vimentin-.9. The phenotypes of CTCs varied even in individuals. In some patients, only onephenotype of CTCs were detected; In others,2to4of all4phenotypes of CTCs weredetected. The numbers of each phenotype were different.10. The Zeb1/Vimentin status of HCC patients detected in tumor tissue was inconcordance with CTCs positive rate（r=0.281，P=0.009）.11. Some Zeb1/Vimentin-positive CTM were detected in some HCC patients.ConclusionIn this study, MACS negative sorting method and CPS1and ASGPR identification significantly increases specificity and sensitivity and it has potential clinical applications.Then it was used for CTC type detection by immunofluorescence staining against variousepithelial and mesenchymal markers. The CPS1+ASGPR/Zeb1/Vimentin multicolorimmunofluorescence method is simple and practical. It is likely clinically useful inisolation and identification of HCC CTCs. The expression of Zeb1and Vimentindiversifies in CTCs and the phenotypes of CTCs varies even in individuals. TheZeb1/Vimentin status of HCC patients detected in tumor tissue was in concordance withCTCs positive rate. CTCs Zeb1/Vimentin detection may help predict patients at risk ofpostsurgical recurrence and perform them further treatment and close follow-up. Thetargeting and blocking of Zeb1and Vimentin will offer new strategies and ideas fortreatment of liver cancer. |
