Role Of Lymphatic Microvessles On Metastasis Of Cholangiocarcinoma

Cholangiocarcinoma is one of the common neoplasms in the field of hepatobililary surgery. Surgery is considered to be the most effective way to treat cholangiocarcinoma. However, the outcome of patients with advanced cholangiocarcinoma is extremely poor even after resection. The presence of lymph node metastasis has been reported to be the worst prognostic factor after resection in most previous studies.The lymphatic vascular system plays important roles in the maintenance of tissue fluid homeostasis, in the mediation of the afferent immune response, and in the metastatic spread of malignant tumors to regional lymph nodes. It consists of a dense network of blind ending, thin-walled lymphatic capillaries and collecting lymphatics that drain extravasated protein-rich fluid from most organs and transport the lymph via the thoracic duct to the venous circulation. Tumor metastasis to regional lymph nodes represents the first step of tumor dissemination in many human cancers and serves as a major prognostic indicator for the progression of the disease. In contrast to the extensive molecular and functional characterization of tumor angiogenesis, little is known about the mechanisms through which tumor cells gain entry into the lymphatic system. The recent identification of lymphatic growth factors and receptors, together with the discovery of lymphatic-specific markers and the development of orthotopic cancer metastasis models, have provided important new insights into the formation of tumor-associated lymphatic vessels and their active contribution to lymphatic tumor spread. An increasing number of clinicopathological studies have shown a direct correlation between tumor expression of the lymphangiogenesis factors VEGF-C or VEGF-D and metastatic tumor spread in many human cancers, including cancers of the breast, lung, prostate, cervix, and colon, providing circumstantial evidence for the involvement of lymphangiogenesis in tumor progression. Firstly, combined 5'-nucleotidase enzyme(5'-Nase) and CD31/type â…£ collengen double immunohistochemical approaches were adopted to distinguish microlymphatics from microvasculars in clinical surgical specimens from patients with cholangiocarcinoma. Meanwille Sections were immunostained by using antibodies to human VEGF-C, VEGF-D, VEGFR-3, or Ki-67. Vessel counts were performed. Reverse transcriptase reaction-polymerase chain reaction(RT-PCR) and Western blot were used to explore the expression difference in mRNA and protein level according to the manufacturer's instructions. Thin sections of clinical surgical specimens were examined with an electron microscope to observe the ultrastructure of lymphatics. Thereafter relationships between expression of VEGF-C/VEGF-D and clinico-pathological characteristics were analyzed. The results show as follow: Functional lymphatics exist in the tumor margin. Lymphatic capillaries contain no smooth muscle and are generally observed in a partially or fully collapsed state intratumor and typically dilated peitumor. lymphatic vessels containing clusters of tumor cells are frequently observed at the periphery of tumors. The total positive expression rate of VEGF-C in all the specimens was 79.7%(47/59). The level of VEGF-C was closely correlated with the degree of tumor aggression, lymph node metastasis and distant metastasis. Thenceforth, an infiltration screening model which utilized the artificial basement membrane and Millipore as the carrier was established. And utilized such model, we successful screened out high and low metastatic QBC939 cell subgroups. Invasive experiments in vitro also proved that the invasive ability of high cell subgroup was stronger than that of the low one. Different expression of VEGF-C between above cell subgroups was measured by immunohistochemstry, RT-PCR and Western blot alone and after mixed cultivation with 3T3 cells, expression of VEGF-C. Both cell subgroups were transplanted into the back skin of the nude mice to investigate the capacity difference of lymphangiogenesis. Results show as follow. Expr...