I-TAC—CXCR3Induced Vasculogenic Mimicry In Hepatic Metastasis Of Colonic Carcinoma

Background1.Explore the predictive transfer mechanisms is a key factor to affect the prognosis of colon cancer liver metastases.Colon cancer is a common digestive malignant tumors.3of the total gastrointestinal tract tumors. In recent years, the majority of patients can be found at an early stage and treated accordingly, even after the early resection of primary tumor, however, about40%of patients with abdominal recurrence and liver metastases in five years, and its incidence is increasing every year. Tsai et al found that colon cancer accompanied by liver metastases liver resection in the same period5-year survival of only16.8%. Chemotherapy (hepatic perfusion chemotherapy) targeting high damage to normal tissue greater long-term survival of patients no significant improvement. Cryoablation and radiofrequency ablation to kill tumor cells, the exact effect, but whether it can prolong the overall survival time, no clear conclusion. That colon cancer liver metastases steps include the degradation of extracellular matrix (ECM), changes in cell adhesion, local invasion of tumor cells, tumor angiogenesis, spread within the tumor cell cycle and immune escape of tumor cells intravascular embolization and tumor cells in the microenvironment of re-growth and other aspects. Looking for more effective interventions methods and explore the prediction of transfer mechanisms is the key to solve the problem.2. Current research status of angiogenesis in liver metastases of colon cancerTumor angiogenesis played a very important role in the process of tumor formation and metastasis,. Tumor of a subset of cells converted to angiogenesis gene phenotype. Angiogenesis began to rapidly generate, the tumor will be further growth and occurrence of metastasis. The Strieter proposed imbalance between the factor and vascular stabilizing factor in the angiogenesis in tumor growth and development process. Tumor cells can be generated by overexpression of one or more of the blood vessels of the adjustment factor, to promote the release of stromal cells containing the angiogenesis protein, to promote host cell to release its angiogenic factors, such as to promote local angiogenesis. The most important factor to promote tumor angiogenesis is vascular endothelial growth factor (VEGF). In colon cancer. VEGF is an important factor in colon cancer liver metastases.In addition to VEGF, platelet-derived endothelial cell growth factor (PD-ECGF) has an important role in colon cancer angiogenesis. PD-ECGF is a pro-angiogenic factor, PD-ECGF may play an important role in the low expression of VEGF-rich angiogenesis in colon cancer.Vasculogenic mimicry is a new tumor microcirculation model, which does not depend on the body’s endothelial cells. In angiogenesis, previous studies of colon cancer liver metastasis focuses on the increase in the density of tumor blood vessels and generation of vascular factors. For the relationship of angiogenesis mimicry and colon cancer metastasis, there is no system research at present.3I-TAC-CXCR3induced angiogenesis research status.The main role of chemokines chemotactic cell migration, cell migration along chemokine concentration increased signal at the source of chemokine. Recent studies have found that chemokines as a key signaling molecule in the tumor microenvironment in tumor invasion and metastasis plays an important role. The first time in1998confirmed that βR-1gene encoding a product of the chemokine I-TAC, coding gene located on human chromosome4q21. Consists of94amino acids (including the signal sequence of21amino acids), and mature I-TAC by73amino acids, the molecular weight of about8300, belong to ELRCXC chemokine family. I-TAC receptor CXCR3, composed of368amino acids, its coding gene is located on human chromosome Xq13. CXCR3molecular structure contains seven sulfhydryl amino acid-rich a-helix transmembrane domains belong to the seven transmembrane transport of G protein-coupled receptor superfamily, heterotrimeric G-proteins to pass the signal. CXCR3. I-TAC receptor, is also of Mig, IP-10receptor. Mainly distributed in Thl cells and B cells, NK cells and EC expression. In angiogenesis, I-TAC can be generated as a vascular or anti-angiogenic factor, and I-TAC, and CXCR3and VEGF mutual synergy. Cancer cells can directly produce I-TAC. and also through the regulation of inflammatory cells around the release of I-TAC for the regulation of angiogenesis. In addition, the apoptosis of CXCR3can also be directly regulated by. Chemotactic factor I-TAC vascular regulatory role with the combination of of CXCR3is associated.At present, the study says that I-TAC-CXCR3not only mediated directional movement of cancer cells, also participated in the penetration of blood vessels, anchor, wear clothing with blood vessels, immune escape, proliferation and angiogenesis. Found in surgical specimens of gastric cancer tumor tissue CXCR3expression was significantly higher than the corresponding adjacent tissues and gastric cancer cell lines CXCR3also increased. Infer that CXCR3and its ligand synergy with adhesion molecules to determine tumor cell invasion and proliferation. The study reported that the renal cell carcinoma and melanoma cells can express CXCR3, and through its ligand mediated directional movement and proliferation of cancer cells. In mouse models, such as raising the level of CXCL9and CXCL10, highly metastatic melanoma cell line B16F10transfer capacity can be increased by three times, the use of antisense RNA to reduce B16F10cells of CXCR3expression, or the use of antibody and CXCL9and CXCL10the role of the cells almost lost transfer ability.Angiogenesis is to generate new blood vessels, repair the damage the blood vessels of the physiological and pathological processes. This process had been pro-angiogenic factors and inhibition of vascular endothelial growth factor regulation.We put forward the following questions:I-TAC-CXCR3whether induced colon cancer liver metastasis and vasculogenic mimicry how to interfere with the colon cancer liver metastases? I-TAC-CXCR3induced vasculogenic mimicry formation is one of the colon cancer liver metastasis mechanism?In summary:(1) We detect I-TAC-CXCR3of colon cancer liver metastasis patients with colon tumors and liver metastases by immunohistochemical methods; by PAS and CD31double staining detection of colon cancer organizations and livermetastatic tumor vasculogenic mimicry expression, and analyze its relationship with colon cancer clinicopathological parameters, and confirmed role of I-TAC-CXCR3and vm in liver metastases of colon cancer;(2) We detect CXCR3and protein expression in cell lines of colon cancer liver metastasis by RT-PCR and Western blot and construct CXCR3plasmid vector, and stably transfected tumor cells, and then detect that I-TAC-CXCR3play the tumor cell proliferation, migration, and invasion a role. If the experiment confirmed that I-TAC-CXCR3and vasculogenic mimicry in differential expression between the liver metastasis group and non-liver metastasis group of colon cancer, and the rate of colon cancer lymph node metastasis, degree of differentiation and clinicopathological parameters of relevance:and confirmed that I-TAC-CXCR3inducing vasculogenic mimicry is one of the mechanisms to colon cancer liver metastases. So the application of I-TAC-CXCR3and vascular mimicry to detect the tumor, we set up a prediction mechanism of colon cancer liver metastases, we may be the early intervention to colon cancer metastases by silence CXCR3in colon cancer and further reduce the transfer rate to provide theoretical support.Objectives1. Clinical researchWe detect I-TAC-CXCR3of colon cancer liver metastasis patients with colon tumors and liver metastases by immunohistochemical methods; by PAS and CD31double staining detection of colon cancer organizations and livermetastatic tumor vasculogenic mimicry expression, and analyze its relationship with colon cancer clinicopathological parameters, and confirmed role of I-TAC-CXCR3and vm in liver metastases of colon cancer;2. Cell researchWe detect CXCR3and protein expression in cell lines of colon cancer liver metastasis by RT-PCR and Western blot and construct CXCR3plasmid vector, and stably transfected tumor cells, and then detect that I-TAC-CXCR3play the tumor cell proliferation, migration, and invasion a role.MethodsClinical research1.Collect22patients who had done with colon cancer and liver metastases resection for colon cancer liver metastasis; select20cases of colon cancer without liver metastasis in the control group;collect the tumor tissue of colon cancer and liver metastasis. All specimens are stored separately.2. Detect the expression of the I-TAC-CXCR3in colon carcinoma and liver metastasis tissue by immunohistochemical methods. 3. Detect expression of vasculogenic mimicry in colon cancer tissue by PAS and CD31double staining methods, PAS stain-positive and CD31antibody tested negative is considered vasculogenic mimicry formation.Cell research1.CXCR3plasmid vector construction and identification, transfecting tumor cells and expression detection:total RNA extracted from the human colon cancer tissue, using RT-PCR method to get the full-length CXCR3cDNA fragments, and stable transfection of tumor cells, after screening by PCR were identified.2.Experimental groups:(1) pEGFP group;(2) CXCR3group;(3) pEGFP+I-TAC group;(4) CXCR3+I-TAC group3.Detect CXCR3mRNA and protein expression levels in cell lines of colon cancer liver metastasis by RT-PCR and Western blot4.Observe the presence or absence of I-TAC to CXCR3cells and pEGFP cell proliferation directed migration and invasion of differences from in vitro cell model. and explore the role of I-TAC-CXCR3to colon cancer liver metastasis cell proliferation, directed migration and invasion.ResultsClinical researchColon cancer liver metastasis group of22patients (each patient take a colon cancer and a liver metastasis lesion tissue), a total of44tissue samples. Colon cancer without liver metastases group of20patients (every patient take a piece of colon cancer tissue), a total of20tissue samples.1.CXCR3expression in42cases of colon cancer tissue,16cases of positive expression of the liver metastasis group, the positive rate of72.72%.6cases of negative expression,7cases of positive expression of without liver metastasis group, the positive rate of35%,13cases of negative expression. Difference was statistically significant (χ2=6.019, P=0.014). 2.I-TAC expression in42cases of colon cancer tissue,19cases of positive expression of the liver metastasis group, the positive rate of86.36%,3cases of negative expression.9cases of positive expression of without liver metastasis group, the positive rate was45.00%,11cases of negative expression. Difference was statistically significant (χ2=8.066, P=0.005)3.CXCR3expression in22cases of liver metastasis group,16cases of positive expression of the colon cancer tissue, the positive rate of72.72%,6cases of negative expression.16cases positive expression with liver metastatic cancer tissue, the positive rate of72.72%,6cases with negative expression. Difference was not statistically significant (χ2==0.000, P=.000)4. ITAC expression in22cases of liver metastasis group,19cases of positive expression of the colon cancer tissue, the positive rate of86.36%,3cases of negative expression,19cases positive expression of liver metastatic cancer tissue the positive rate of86.36%,3cases with negative expression. Difference was not statistically significant (χ2=0.000, P=1.000)5. VM expression in42cases of colon cancer tissue, VM14cases positive expression of the liver metastasis group, the positive rate was63.63%.8cases with negative expression.5cases of positive expression in the colon cancer tissue of the without liver metastasis group, the positive rate was25.00%,15cases with negative expression. Difference was statistically significant (χ2=6.313, P=0.012)6. Analysed42patients test results, ITAC were all positive in23cases of CXCR3-positive cases:CXCR3were positive in19cases of VM-positive cases.Statistical results match correlation analysis of the data,ITAC and CXCR3positive expression was positively correlated (r=0.614,p<0.001), vasculogenic mimicry and CXCR3both positive expression was positively correlated(r=0.637, p<0.001). 7. Relationship of CXCR3expression and clinical pathological features of colon cancer:CXCR3-positive expression was mainly located in the cell membrane, a few expressed in the cytoplasm and nucleus. According to CXCR3positive cells rate and the color intensity of the colon cancer samples, the results showed that CXCR3expression in colon carcinoma is different, CXCR3was highly expressed in the colonic carcinoma, which was related to the metastasis of the liver and the lymph node. However, higher expression of CXCR3had nothing to with the patient’s age, gender and tissue histologic differentiation of colonic carcinoma.Cell research1.CXCR3protein expression:positive CXCR3gene expression in colon cancer liver metastases of primary cultured cell lines, and can be detected at the protein level., three groups of cells (cell group of PGFP-CXCR3; pGFP; the untransformed QBC939cells) had positive expression of CXCR3gene at the RNA level, and the rules of RNA is:cell group pGFP-CXCR3> cell group pGFP> cell group is not transformed QBC939cells. But the difference was not large. the amount of protein of CXCR3expression is likely regulated by translation.2.I-TAC-CXCR3paste the impact of the wall proliferation (WST-1method) of tumor cells in vitro.The same conditions in vitro culture, cell group pGFP-CXCR3in vitro proliferation of adherent capacity is stronger in the three cell groups; followed by an untranslated QBC939cells; the worst conversion pGFP cell group.3.I-TAC-CXCR3to tumor cells in vitro the influence of migrating ability.At the same time have CXCR3protein and ITAC protein in vitro culture conditions, QBC939in vitro migration capacity strongest, in the lack of CXCR3and ITAC protein in vitro culture conditions, QBC939cells in vitro migration capacity weakest 4.Transwell migration assay:pGPF-CXCR3colon cancer cell groups occurred in the role of chemokines in vitro migration of cells significantly increased, pGFP colon cancer cell group in vitro migration was significantly less.ConclusionsClinical research1.1-TAC-CXCR3is highly expressed in colon cancer and liver metastases, support the I-TAC-CXCR3and colon cancer liver metastasis have correlation. I-TAC-CXCR3signaling pathway may play a role in the process of colon cancer liver metastasis.2.Vasculogenic mimicry in colon cancer tissue. Vasculogenic mimicry in highly expressed in colon carcinoma of the liver metastasis group, indicating that vasculogenic mimicry and liver metastasis have correlation.3.CXCR3and vasculogenic mimicry positive expression are positively correlated,I-TAC-CXCR3may induce vasculogenic mimicry formation.Cell research1.In this study, from the original generation of colon cancer liver metastases cell lines CXCR3gene expression has been detected on RNA and protein levels.2.Using the SMART RACE method submitted CXCR3gene, full-length cDNA and construct the vector pGFP-CXCR3and pGFP. Successful conversion QBC939cells get QBC939cell lines pGFP-CXCR3and cell lines pGFP-CXCR3.3.Real-time PCR assay results showed that three groups of cells have the CXCR3gene expression in the RNA level, and the rules of RNA is:cell group CXCR3-pGFP> cell group pGFP> QBC939cell group is not transformed cells. But the difference was not large. the amount of protein of CXCR3expression Is likely regulated by translation.4. WST-1method to detect cells in vitro stick wall proliferation ability.The same conditions in vitro culture, cell group pGFP-CXCR3in vitro proliferation of adherent capacity is stronger in the three cell groups; followed by an untranslated QBC939cells; the worst conversion pGFP cell group. Chemokine receptor CXCR3play a positive role in the proliferation of adherent cells.5. Transwell experiments show that chemotactic factor receptor on the invasion ability of CXCR3cells is function.6.Another set of experiments showed that at the same time have CXCR3protein and ITAC protein in vitro culture conditions, migration in vitro QBC939best, the lack of CXCR3and ITAC protein in vitro culture conditions, QBC939cells in vitro migration the weakest. In this study, the interaction between the receptor protein CXCR3and chemokines ITAC to migratory ability of cells has a positive effect from certain angles.summaryIn this experimental study, we applied immunohistochemical methods to determination of I-TAC-CXCR3was highly expressed in colon cancer and liver metastasis tissue, the application PAS/CD31double staining determination vasculogenic mimicry in colon tissue of the liver metastasis group into a high expression, while cultured colon cancer liver metastasis cell lines from in vitro cell model confirmed that I-TAC-CXCR3on colon cancer cell proliferation, directed migration and invasion ability. The data obtained through statistical processing, analysis, and thus we believe that the I-TAC-CXCR3by vasculogenic mimicry-induced colon cancer hepatic metastases is one of the mechanisms. So, expression changes of I-TAC-CXCR3and vasculogenic mimicry in tumor tissue was detected, we set up colon cancer liver metastasis prediction mechanism, we may be the early intervention to colon cancer metastases by silence CXCR3in colon cancer and further reduce the transfer rate to provide theoretical support.