Role And Mechanism Of CCL21-CCR7Inducing Vasculogenic Mimicry In The Micrometastasis Of Postoperative Hilar Cholangiocarcinoma

Background:Hilar cholangiocarcinoma is primary epithelial Carcinoma from to the common hepatic duct to the left and right hepatic ducts, of the liver, and is58-75%of all the extrahepatic bile duct Carcinoma. Due to size, and vascular space closely as well as characteristic of tumor infiltrative growth, it has a low rate of surgical resection, radical resection rate is lower. However, even after radical resection (cutting edge and surrounding tissue no residual tumor cells by routine pathological examination), recurrence rate remains high and mainly hepatic portal space metastasis. Therefore we speculate micrometastasis may be the first important, and a number of studies have shown that tumor micrometastasis is independent factors for prognosis. So reducing micrometastasis is key factors for improving hilar cholangiocarcinoma cure rate.Micrometastasis is phenomenon that routine clinical pathology method could not be checked out and transfers by non-hematological malignancies, and leaving the microscopic examination of metastatic (<2mm) deposits of cancer cells, and has vascular violations after evading immune surveillance and become visible to the naked eye lesions, often without any clinical manifestations. Vasculogenic mimicry is important and different tumor micrometastasis way of tumor angiogenesis, does not depend on tumor microcirculation pattern by endothelial cells. Thelen found tumor angiogenesis was related to lymph node metastasis and local recurrence, and affected survival rates after resection. Aishima’s research findings showed tumor microvessel density increase promoted lymph node metastasis and blood vessels infiltration. Vasculogenic mimicry is basis of formation mosaic vessels and endothelial vessles.SLC was first reported by Nagria in1997, belonging to the CC Chemokine. Human SLC cDNA sequences including847bases, encoding134amino acid residues,21%～33%homology with other Chemokines. SLC gene map in9p13, and has4Exon and3intron. SLC is produced by T-lymphocytes and endothelial cells, high expression in secondary lymphoid tissue, particularly the high endothelial venule and lymph node T-cells. SLC includes CCL21and CCL19, and CCL21high expression in lymph nodes show CCL21plays an important role in lymphocyte homing. CCR7, high affinity receptors by SLC, found in the EB virus-infected B-cells by Yashida in1998, and it has378-containing amino acid residues, gene map in17q12～Q21.2. It expressed in Mainly naive T-cells, B-cells and dendritic cell, memory T-cells, NK cells and NKT cells also express CCR7. High level CCL21expression in tumor Common transfer parts shows that CCL21and CCR7are likely involved in tumor metastasis and invasion, and CCR7expression and the dose-dependenence to CCL21may determine the direction and location of tumor metastasis. CCL21-CCR7has a certain role in Lymphoma tumor homing, angiogenesis and nerve invasion, And they can control other Chemokines secretion.We proposed high hilar resection and portal parenchyma-enterostomy method to expand the scope of surgical resection, as far as possible reduce the occurrence of micrometastasis; We detected CCR7expression in hilar cholangiocarcinoma tissue and cells, on this basis,(1) Clinical study: high hilar resection and portal parenchyma-enterostomy method’s anatomic basis, compared to the advantages and disadvantages from traditional operation, reducing micrometastasis and the need for improved vision.(2) Basic research:existence of vasculogenic mimicry of hilar cholangiocarcinoma? does CCL21-CCR7axis induce vasculogenic mimicry to promote hilar cholangiocarcinoma micrometastasis? How about mechanism of formation of vasculogenic mimicry induced by CCL21-CCR7? In short, through a combination of basic and clinical study, we try on new method for blocking hilar cholangiocarcinoma micrometastasis, increasing radical resection rate, reducing postoperative recurrence rates, extending patient survival time, improving quality of life. Part IHigh hilar resection and portal parenchyma-enterostomy for Bismuth type IV hilar cholangiocarcinomaObjectiveTo investigate clinical result of hepatectomy and porta—enterostomy in thetreatment of Bismuth type IV hilar cholangiocarcinoma.MethodsThirty-two cases of Bismuth type IV hilar cholangiocarcinoma treated from March2004to June2011in our hospital were reviewed retrospectively. They were divided into2groups by operation method:new surgical group(N, n=16) and tranditioanal surgical group (T, n=16). New surgical approach group by high hilar resection and portal parenchyma-enterostomy and tranditioanal surgical approach group by hepaticocholangiojejunostomy after hepatic duct shape, all of these are radical resection.Then operation time, blood loss, inpatient days, Complications, post-operative quality of life and survival rate were comparatively analyzed.ResultsCompared to the traditional surgical group, operation time\inpatient days\number With liver or segmentectomy in the new surgical group decreased significantly; There were no significant difference in blood loss\post-operative quality of life\Complications, but bile leak rate was lower in new surgical group than those in traditional surgical group and bile duct infection rate higher in new surgical group than those in traditional surgical group; The1-,3-and5-year survival rates was higher in new surgical group than those in traditional surgical group. ConclusionWith accurate high hilar resection and portal parecnchyma-to-enterostomy the patients considerably benefit from the preservation of liver parenchyma and patent biliary drainage and radical resection. So the new technique prolongs the survival time and enhances the quality of life of the patients.SignificanceHigh hilar resection and portal parenchyma-enterostomy made the operation procedure simple, reduced the touch metatasis and improved resection rate and survival rate. So the new surgical approach Is worth promoting. Part IIExpression of chemokine receptor CCR7and vasculogenic mimicry in hilar cholangiocarcinomaObjectiveTo investigate expression of chemokine receptor CCR7and vasculogenic mimicry in hilar cholangiocarcinoma tissue\cell line and relationship with clinical pathological parameters.MethodsThe expression of chemokine receptor CCR7and vasculogenic mimicry was tested by immunohistochemical and dual staining method respectively in63hilar cholangiocarcinoma specimens and20normal bile duct specimens, and had correlation test with clinical pathological parameters. The expression of chemokine receptor CCR7in cell line was tested by RT-PCR and Western blot; Vasculogenic mimicry was tested in cell line by three-dimension cell culture.ResultsCCR7expression was positive in79.4%(50/63) of hilar cholangiocarcinoma specimens, in only10%(2/20) of normal bile duct specimens. There was25Vasculogenic mimicry formation in63hilar cholangiocarcinoma specimens, and zero in normal bile duct specimens; All the poorly differentiated carcinoma were positive in Vasculogenic mimicry, and5cases were positive in25middle differentiated carcinoma. Expression of CCR7and Vasculogenic mimicry were relevant to lymph node metastasis and differentiation (p<0.05), but not to age, gender, tumor size, histological types and liver resection (p>0.05).4significant factors (CCR7/Vm/lymph node metastasis/differentuation) were related with survival rate by univariate analysis, whereas in multivariate analysis, only CCR7retained the prognostic significance. CCR7is Consistent with tissue detection, CCR7expression was positive in QBC939cell line; and QBC939cells can formate vascuogenic mimicry by three-dimension cell culture.ConclusionCCR7and vasculogenic mimicry expression were highly positive in hilar cholangiocarcinoma tissue and cell line, and they had positive correlation each other. Expression of CCR7and Vasculogenic mimicry were relevant to lymph node metastasis and differentiation, but not to age, gender and tumor size, histological types and liver resection, and CCR7was independent factor with survival rate.SignificanceTo show Vasculogenic mimicry formation was very important in hilar cholangiocarcinoma micrometatasis; CCR7can induce micronetatasis in hilar cholangiocarcinoma by Vasculogenic mimicry and lymphatic metastasis. Part IIICCR7and CCR7-short hairpin RNA Expression Vectors Construction, transfection and Screening of effector cellsObjectiveTo construct CCR7and CCR7shRNA eukaryotic expression vectors to transfect into QBC939cells in order to get CCR7high, regular and low expression stable cell.MethodsAfter Extracting total RNA from QBC939cells, CCR7product was got through CCR7primer by RT-PCR; By enzyme reaction and access to Insert DNA and Vector DNA, fluorescent CCR7plasmid was got by connection and transformation. Similarly, under the principle of designed shRNA designed shRNA-specific sequence and got shRNA by Lentiviral expression vector construction, then transcripted shRNA-CCR7and green fluorescent protein (green flurescence protein, GFP) together by the RNA polymerase Ⅲ promoter U6to manipulate a small hairpin siRNA expression in QBC939cells. Through three plasmid transfected293T cells, carrying different CCR7lentiviral expression plasmid were producted. Then hese venom directly infected with QBC939cells, Finally with an adequate dose of Puromycin killed non-transducted cells.ResultsBy the CCR7-pEGFP plasmid DNA sequencing, sequencing results was consistent with experimental design&synthesis of CCR7target gene sequence; CCR7-shRNA plasmid DNA sequencing results and experimental design&synthesis of shCCR7target gene sequences was exactly the same. pEGFP cells, CCR7-pEGFP cells and CCR7-shRNA-pEGFP cells were got by lentivirus transfection. Compared to the control group, CCR7expression markedly enhanced in CCR7-pEGFP group; CCR7-shRNA-pEGFP silented CCR7expression by RT-PCR and Western-Blot detection.ConclusionSuccessful synthesis of fluorescent-labeled CCR7high expression plasmid and CCR7-shRNA plasmid; pEGFP, CCR7-pEGFP and CCR7-shRNA-pEGFP three cell lines were got through lentivirus transfection finally.SignificanceTo provide assurance for next research on CCR7high, normal and low expression’s effect on VM formation, cell proliferation, migration and invasive in hilar cholangiocarcinoma. Part IVRole and mechanism of vasculogenic mimicry induced by Chemokine receptor CCR7in hilar cholangiocarcinoma micrometastasisObjectiveTo explore Chemokine receptor CCR7promoting QBC939cell proliferation, migration and invasion by inducing vasculogenic mimicry formation as well as the role of CCR7signaling pathway.MethodsAccording to culture medium containing CCL21or not, three different CCR7expression cell lines were divided into six groups:1) pEGFP group2) CCR7-pEGFP group3) CCR7-shRNA-pEGFP group4) pEGFP-CCL21group5)CCR7-pEGFP+CCL216) CCR7-shRNA-pEGFP+CCL21group. Vasculogenic mimicry in QBC939cells were tested by three-dimensional cell culture; cell migration capabilities of groups of cells were detected by scratch test and Transwell chamber; Boyden chamber detecting CCL21-CCR7impact on the invasive ability of QBC939cells in vitro; Application of MTT assay for detecting effect of CCL21-CCR7on proliferation of QBC939cells. Finally detecting expression of CCR7and related6gene Twist1\GAPDH\IL-8\VEGF\PDEF\PDCD4in three groups of cells by Real time PCR.ResultsCompared to control group, VM formation ability and cell proliferation were strengthened obviously in CCR7high expression group and reduced in CCR7silence expression group; VM and cell proliferation did not changed after adding CCL21in the three groups. Detecting CCL21-CCR7impact on the migaration and invasive ability of QBC939cells in vitro by scratch test, Transwell and Boyden chamber got consistent results:compared to control group, CCR7expression group crawling ability and number of film cells obviously increased in the CCR7high expression group and reduced in CCR7silence expression group; After joining CCL21crawling ability and number of film cells increased in the control group and CCR7high expression group, and no change in CCR7silence expression group. qPCR detection by applying CCR7and Twist1\GAPDH\IL-8\VEGF\PDEF\PDCD4primers showed, Twistl gene in three groups changed in trend consistent with changes of CCR7, CCR7high expression group also Twistl high, CCR7silence group Twistl gene expression is also low.ConclusionDifferent expression of CCL21-CCR7had a effect on the formation of vasculogenic mimicry, so bile duct cancer cell proliferation, migration and invasion changed;CCR7’s effect on VM formation, cell proliferation, migration and invasion may be achieved through Epithelial-mesenchymal transformation of Twistl.SignificanceTwistl inducing epithelial-mesenchymal transformation by interaction of CCL21/CCR7may be one of the important mechanisms for forming VM to promote hilar cholangiocarcinoma micrometastasis. While the active primary tumor treatment, application of interaction between Chemokine and its receptor blockers, plus treatment of blockers such as VM, Twistl in its micrometastasis signaling pathways together, may be more efficient and significantly reduce micro-metastasis after operation of hilar cholangiocarcinoma, then improve patients survival rates and prognosis.