The Study Of Slug In Cancer Stem Cell Phenotype And Vasculogenic Mimicry Formation In Hepatocellular Carcinoma

[Purposes]vasculogenic mimicry (VM), in which tumors can feed themselves, has been reported is formed by tumor cells. The presence of VM was associated with a high tumor grade, invasion and metastasis, and short survival. Slug (SNAI2) was reported to be an essential transcription factor of EMT. Slug have been shown to not only induce EMT, but also have associated with CSC "sternness" behavior. CSCs have been shown to promote tumor angiogenesis and have the ability of transdifferentiation into endothelial cells. However, the relationship of EMT, CSCs phenotype and VM in HCC is currently unknown. Clinical specimens of human hepatocellular carcinoma of113patients with complete follow-up data were studied in.this study to observe the relationship of VM and Slug. Analyze the relationship between Slug and E-cadherin, vimentin,VE-cadherin,VEGF, CD90,CD133et al, thus the function of Slug associated with recurrence and metastasis, prognosis, CSCs phenotype and VM will be known. The resultes will be proved again in vivo and in vitro. Expecting to provide a new target for the treatment of HCC.[Methods]1、 Resected specimens of113patients identified as hepatocellular carcinoma with complete follow-up data were obtained from General Hospital and Cancer Institute and Hospital of Tianjin Medical University to analyze the relationship of Slug and age, gender, recurrence and metastasis, and clinical stages between Slug positive group and Slug negative group. Kaplan-Meier survival analysis was performed to see if there is significant difference in the prognosis between the two groups.2、Select several HCC cell lines. Slug expression plasmid and shRNA plasmid were constructed. Cells were transfected to acquire up-regulation model and down-regulation model. Western blot was used to identify the transfection efficiency. Transwell invasion assay were performed to evaluate the effect of Slug on cell motility and invasion; three-dimensional matrigel culture was used to further analyze the effect of Slug on angiogenesis of tumor cells; chromatin immunoprecipitation assay and cDNA microarray analysis were performed to investigate the singaling pathway regulated by Slug.3、Building of animal model:Male BALB/c nude mice,5weeks of age, were injected the HepG2and HepG2-Slug cells.5X106viable cells/0.1mL of PBS were injected into the armpit of20mice. For30days the mice were monitored and tumor sizes were measured weekly using a caliper. The tumor volume (TV) was calculated by the following formula:TV=1/2×axb2(in which a is the length and b is the width of tumor). Compare the formation of VM, the expression of E-cadherin, vimentin,VE-cadherin,VEGF, CD90,CD133et al in Slug-positive group and Slug-negative group.[Results]1-. Expression of slug in correlation with VM in human HCC tissueBy CD31and periodic acid-Schiff (PAS) histochemical and immunohistochemical double staining, typical microvessels showed positive reaction for CD31on their luminal surface and PAS-positive reaction in their wall. CD31-negative, PAS-positive vascular-like patterns containing red blood cells, which formed by HCC cells, were deemed VM. CD31/PAS double staining shows that VM present in17.7%(20/113) HCC patients. The highest VM area was identified and individual VM channel counts were made on a200x field. By VM channel counting, the median value showed4.20±0.80in slug positive-expression and0.18±0.12in slug negative-expression. There were significant differences between the two groups (t=6.662, P=0.000). Therefore, the presence of VM was closely associated with slug positive-expression. slug positive tumor cells showed close relationship with vascular vessel formation. Slug positive tumor cells either could form vasculogenic mimicry or involved in mosaic vessels with endothelial cells, suggesting slug played an important role in tumor vasculature. Hepatocyte antibody was used to demonstrate the cells forming VM were HCC cells. HCC cells with hepatocyte positive formed VM, and IHC double staining showed that hepatocyte+/slug+HCC cells were part of vascular vessels.Vascular endothelial (VE)-cadherin had been identifed as a sign in VM formation. In this study, IHC shows that48(42.5%) of113cases showed VE-cadherin expression in HCC specimen. By statistical analysis, VE-cadherin expression was positively related with slug expression (r=0.205, P=0.030). vascular endothelial growth factor (VEGF) expression present in61.1%(69/113) HCC samples. VEGF showed a higher expression in slug positive than in slug negative (χ2=4.429, P=0.035) and displayed significant correlation with slug overexpression by statistical analysis (r=0.198, P=0.036).2、Expression of slug in correlation with metastasis and prognosis in HCC patientsThe clinicopathological data in patients with slug positive (n=39) were compared with those with slug negative (n=74) in HCC. Among all factors (age, gender, differentiation grade, stage and metastasis) compared, metastasis was significant different between groups with slug+and slug" by binary logistic regression analysis (P=0.036). The comparison of slug expression was made between the metastasis (47cases) and nonmetastasis groups (66cases). The positive rate of slug was46.8%(22/47) in the metastasis group, higher than that of nonmetastasis group (17/66,25.8%). The difference was statistical significant (χ2=5.382, P=0.020). Survival analysis indicated that patients with slug positive in HCC were significantly associated with poor overall survival. The mean (95%CI) overall survival time was34.311(27.084-41.538) and44.721(38.192-51.251) months respectively for patients with and without slug positive expression in HCC (P=0.025).3、EMT could induced by Slug Slug has been considered as EMT inducer and E-cadherin inhibitor, then E-cadherin and vimentin expression were examined. Statistically significant correlations were found between E-cadherin, vimentin and slug expression. Slug overexpression significantly correlated with E-cadherin reduced expression and vimentin increased expression. By IHC double staining, we found that slug+cells lost E-cadherin expression in the cell membrane, and slug-cells could express E-cadherin in the cell membrane.4、Expression of slug in correlation with cancer stem cell phenotypeCD133and CD90are identified as specific antigenic markers for HCC stem cells.By statistical analysis, there is correlation between slug and CD90expression (r=0.207, P=0.028). More patients with high slug expression displayed high CD90expression (30.8%,12/39), whereas low slug expression showed1.4%(10/74) cases with CD90positive expression. Therefore, our data suggest that slug has a close relationship with CSCs phenotype CD90. Meantime, IHC double staining (CD90/hepatocyte) was used to testify CD90positive cells were HCC cells. Importantly, CD90+/hepatocyte+HCC cells were shown to be involved in VM formation. Slug coexpression with CD90and CD133in HCC cell. We observed CD133+or CD90-HCC cells were involved in VM formation directly. It implied that HCC cells with CSCs marker CD133/CD90expression could form VM channel.The expression of pluripotency maintaining factors (sox2, nanog, oct-4) which are involved in specification and maintenance of cancer stem cells were examined by immunohistochemistry. Positive cells were indicated by the presence of brown staining in the nucleus. Sox2and Nanog were detected in29.2%and13.3%of hepatocellular cancer tissues; whereas there was lack of Oct-4expression in all the113HCC cases. There was significant correlation between slug and sox2expression (r=0.230, P=0.014), slug and nanog expression (r=0.210, P=0.026). We observed that sox2positive tumor cells could form blood vessels. By IHC double staining, we could observe the co-localization of slug and sox2. Sox2+/slug+HCC cells were involved in VM formation. The experimental results are verified in vivo and in vitro. [conclusions]1、There was vasculogenic mimicry in hepatocelluar carcinoma, which make tumor cells acquire blood supply to sustain growth and metastasize by this special microcirculation pattern. VM was mainly existed in the poorly differentiated and high-malignant HCCs who belonged to solid and poorly differerntiated type, suggesting that VM was associated with malignant biological behaviors of HCC.2、EMT regulation factor Slug was in correlation with metastasis and VM in HCC patients. However, there was no significant different between the expression of slug and age, sex, clinical stage, pathological classification, etc. The present of Slug is associate with the poor prognosis and shorter survival time.3、Slug overexpression was not only related to EMT, but also related to CSCs phenotype. A significant positive correlation between slug expression and SOX2、 nanog、CD90、CD133、VEGF、VE-cadherin、Vimentin was also observed. the expression of SOX2and nanog could reprogram HCC cells to a pluripotent stem cell state. In addition, ectopic slug overexpression in vitro and in vivo could induce EMT, VM and CSCs phenotype in HCC cells.