Influence Of Human Rectal Cancer Homogenate Supernatant On Dentritic Cells Endothelialization

BackgroundDendritic cell is the most potent professional antigen present cell, is also an initiator of immunological reaction. It plays a very important role in induction of tumor immunological response. In vitro, tumor can elude by secreting immuological suppressing factors in creating special microenvironment to interference function of dendritic cell. Exploring the function of dendritic cell in tumor microenvironment and its role is a new studying hotspot. The latest finding indicates that tumor-infiltrating dendritic cell precursors may endothelialize under the influence of VEGF-A, to participate in tumor neovasculogenesis, to initiate proliferation, diffuse, metastasis of tumor.ObjectiveWe want to study the function and role of dendritic cell in rectal cancer micrienvironment. We explore the phenomenon of DC endothelialization by inducing DC derived from human peripheral blood by rectal cancer homogenate supernatant. We want to find a new mechinism of tumor neovascularization in order to find a new therapeutic target and way of tumor therapy.Methods1,Observe the expression of S-100 protein on tumor microvessel byimmmohistological chemistry(IHC)We observe the expression of S-100 protein on tumor microvessel by SABC immmohistological chemistry in rectal cancer tumor slides.2,ELISA of tumor homogenate supernatant Transduce the freshly freezed tumor tissue into homogenate and use homogenate supernatant as the revulsant, check the difference of the content of VEGF-A between cancer and peri-cancer homogenate supernatant by ELISA.3,Tumor homogenate supernatant induce DC endothelializationWe induce monocytes derived from peripheral blood into dendritic cells. Add homogenate supernatant into cell medium during immature condition of dendritic cell in order to induce DC endothelialization. As usual, the first cultured 7 day is in immature condition. We devide into 4 different experiment groups: 3^rd cancer supernatant added group; 3^rd peri-cancer supernatant added group; 7 cancer supernatant added group; 7^th peri-cancer added group, as the same time, non-supernatant added group as control.4,Cheak the expression of CD1a/CD31,CD1a/CD34 on cultured cells by FCMFACS Calibar check, analyze checked results by Cellquest analytic software.5,Observe the expression of vWF on cultured cell by immunocytochemistry(ICC)Smear single cell suspension on slides and observe the expression of vWF by biomicroscope.6,Statistical analysisAll data are expressed as mean value±S.D. and analyzed by statistical software SPSS 10.0.The level of statistical significance was set at 0.05.Results1,Demonstration of result from immunohistological chemistry(IHC)We find the expression of S-100 protein on tumor microvascular area. Because the level of expression is too low, we don't analyze statistically.2,ELISA of VEGF-A in homogenate supernatantCompared with peri-cancer supernatant, the level of VEGF-A in cancer supernatant is much higher. The difference is statistically significant.3,The dynamic expression of CD31,CD34,CD1aWe detected the dynamic expression of CD31,CD34,CD1a at different time: 1^st day,3^rd day, 7^th day, 10^th day, 14^th day. The result implicated that the three detected results of CD31,CD34 is reduced increasingly; the three detected results of CD1a is upregulated increasingly during 1-7 day, and don't change significantly during 7-14day.4,Inducing effect of homogenate supernatant in DC endothelializationCultured cells of different groups were totally harvested at 14^th day. Molecular phenotype was detected by FCM.Result from 3^rd day added groups: compared with control, the expression level of CD1a/CD34,CD1a/CD31 of cancer and peri-cancer supernatant were higher, and the difference is statistically significant(p < 0.05).Result from 7^th day added groups: compared with control, the expression level of CD1a/CD34,CD1a/CD31 of cancer and peri-cancer supernatant were higher, and the difference is statistically significant(p < 0.05).The expression level of CD1a⁺/CD34⁺, CD1a⁺/CD31⁺ of 3^rd day added groups is higher than that of 7^th day added corresponding groups.The difference is statistically significant. The expression level of CD1a⁺/CD34⁺, CD1a⁺/CD31⁺ of cancer supernatant added gruops is slightly higher than that of peri-cancer supernatant added corresponding groups, but the difference is not statistically significant(p > 0.05).5,Expression of vWF.Cultured cells of the groups of cancer and pericancer homogenate supernatant began to express vWF, a molecular marker of endothelium cell. Compared with control, the difference is statistically significant. but the difference between cancer and pericancer supernatant is not statistically significant.Conclusion1,We find that there is DC of S-100 protein tagged on tumormicro vascular wall.The phenomenon indicates that tumor infiltrating immature dendritic cell transformed into tumor microvascular area.2,The content of VEGF-A in cancer supernatant is much higher than that ofpericancer supernatant.3,In vitro, tumor homogenate supernatant can induce DC endothelialization. It begin to express vWF, and transform into CD1a⁺/CD34⁺ or CD1a⁺/CD31⁺ endothelium-like cells, contribute to tumor neovasculogenesis.