β-catenin Phenotypic Characterization Of Circulating Tumor Cells And Its Clinical Significance In Hepatocellular Carcinoma

Objectives:Circulating tumor cells (CTCs) plays a important role in the hematogenous metastasis of malignant tumors. Different phenotypes of CTCs may carry different biological information due to the existence of tumor heterogeneity so that molecular phenotypic characterization of CTCs will be able to help us understand the essential feature of the tumor much better. Beta-catenin is an important component of Wnt signaling pathways with a activation state, plays an important role in the development of varies malignant tumor. Sorafenib is the only drug that has been approved to the treatment of advanced hepatocellular carcinoma (HCC). However, not all of the patients have impressed response, the resistance rate of sorafenib is actually quite common. Indeed, individualized treatment is particularly important. Existing related research has built method for guiding sorafenib individualized treatment by CTCs in HCC. It has been reported that sorafenib can inhibit Wnt signaling pathways in hepatocellular carcinoma. Therefore, we try to detect the beta-catenin molecular phenotypic of CTCs, aimed at exploring the potential of guiding sorafenib individualized treatment in patients with HCC.Methods:1. The expression of beta-catenin in different tumor cell lines including QGY-7701、 SMMC-7721、HepG2、Hep3B、Huh-7、LM3、WRL-68、LX2、SU-DHL-4 by Western Blot method.2. The methodology of four-color immunofluorescence staining technique is used to detect the beta-catenin phenotypic in CTCs.(1) Combined the sorting strategy of MACS with Ficoll-Paque PLUS liquid separation density gradient centrifugation, get rid of PBMCs from the blood sample. Spiked the liver cancer cell line QGY-7701 into blood sample to establishe the CTCs model.(2) Four-color immunofluorescence staining was applied to phenotypic characterization of HCC CTCs for detection the expression of beta-catenin.(3) Using the aforementioned four-color immunofluorescence staining method to detect CTCs in blood samples from 35 cases of HCC patients and their beta-catenin expression. As a negative control, included 10 cases of healthy volunteers into testing.(4) Corresponding paraffin tissue sections of 35 HCC patients were examined by immunohistochemistry of beta-catenin expression. Compared the expression of beta-catenin in CTCs with tissues for correlation analysis.3. Expand the testing samples, included in the test of 93 patients with HCC for count of CTCs and beta-catenin expression.12 patients accepted the sorafenib treatment, compared the number of CTCs and beta-catenin expression changes before and after treatment. Collect the related clinical data and follow-up information, making statistics for preliminary assessment of HCC CTCs count and beta-catenin molecular classification in guiding sorafenib individualized treatment application potential.Results:1. Almost all of the cell lines, including the whole hepatocellular carcinoma cell lines, hepatic stellate cell line LX2 and normal cell line WRL-68, have a variety expression level of beta-catenin except the lymphoma cell line SU-DHL-4. The expression level of beta-catenin in hepatocellular carcinoma cell line QGY-7701 is much more than others.2. The four-color immunofluorescent detection model of CTCs, which established by the cell-spiking experiment with several HCC cell lines, can accurately detect the beta-catenin expression differences between different liver cancer cells.3. The four-color immunofluorescent detection model of CTCs can accurately detect the beta-catenin expression differences between different HCC CTCs. In 35 cases of hepatocellular carcinoma patients,88.6%(n=31) cases were detected with the existence of CTCs. The count of CTCs per 5 mL peripheral blood samples has a average of 19±11, ranging from 2 to 51. None of 10 healthy volunteers could detect out any CTCs. In those cases with the present of CTCs, beta-catenin positive CTCs were detected in 80.6%(25/31) patients. The rate of beta-catenin positive CTCs ranged from 0% to 100%. Different beta-catenin phenotype of CTCs can be detected in the same patients. Total number of CTCs was 575,384 CTCs were identified to be positive of beta-catenin expression, with a total positive rate of 66.8%.4. Correspondingly. β-catenin were detected in tissue specimens that 85.7% tumors were classified as β-catenin+. The beta-catenin widely located in cell membrane and cytoplasm,63.3% cases (19/30) exhibit nuclear accumulation.5. The molecular classification of CTCs and tissue specimens was concordant in 90.3%(28/31) patients. The statistical analysis showed that the two results from different samples are basically consistent and have correlation between each other. Therefore, we believe that CTCs samples can accurately represent beta-catenin phenotype characterization instead of tissue samples.6. Expand the test sample, a total of 93 cases were incorporated into detection. CTCs count ranged from 2 to 61, and were detected in 89.2%(83/93) patients with an average number of 24±15.78.3%(65/83) patients were classifie β3-catenin+. A total of 2026 CTCs were detected in 83 HCC patients,64.8%(1312/2026) were beta-catenin positive CTCs. In addition, we found the existence of CTM in one case.7. We took the clinical data statistics of 83 HCC patients and analyzed the relationship between the CTCs beta-catenin phenotype with clinical features. The results showed that the beta-catenin phenotypic of CTCs has correlation with PVTT that P-catenin+patients are more likely to form PVTT.8. A total of 12 patients received sorafenib monotherapy. The majority of patients suffered varying degrees of decline in CTCs counts after two weeks of treatment, from 43±14 to 32±15 (P=0.077). Only one patient appeared the opposite result. The beta-catenin positive CTCs counts pre- and post-treatment has no obvious difference (28±14 vs 25±15, P=0.610). The beta-catenin negative CTCs counts declined significantly after treatment (15±9 vs 7±6, P=0.020). Compared with beta-catenin positive CTCs counts, the rates of decline in beta-catenin negative CTCs is much more evident (52.3% vs 10.8%, P=0.000).Conclusions:The four-color immunofluorescence staining technique we established in this study was convenient and practical, which can be used to beta-catenin phenotype identification in HCC CTCs. CTCs samples can accurately represent beta-catenin phenotype characterization instead of tissue samples. CTCs with various expression of beta-catenin exhibited heterogeneity among different patients and even in the same patient. The β-catenin+ patients are more likely to form PVTT. The current results indicate that beta-catenin phenotype identification in HCC CTCs could be used as therapeutic biomarkers to guide the individualized therapy, real-time dynamic monitoring and evaluation of curative effect for HCC patients undergoing sorafenib treatment. The β-catenin- patients and the β-catenin+ patients which have low rate of beta-catenin positive CTCs can be actively recommend using sorafenib treatment.