| Antitumor angiogenesis therapy has many theoretical advantages over traditional therapy.For example,drugs can directly act on genetically stable endothelial cells,avoid drug delivery through vascular endothelium and reduce drug efficacy,and can greatly improve drug resistance.However,this treatment still has many disadvantages,such as low specificity,obvious systemic toxic and side effects and slow response.Currently,a variety of known tumor vascular endothelial cell markers such as CD34,CD31 and VEGF are also expressed in normal endothelial cells,which will reduce the targeting of tumor therapy.Therefore,the discovery of tumor endothelial cell specific marker molecules is of great significance.In 2000,St Croix et al.[1] found the TEMs family of tumor endothelial cell surface markers in colon cancer tissues for the first time,among which TEM8 was significantly expressed in colon cancer endothelial cells,but hardly expressed in normal vascular endothelial cells.Subsequent studies have confirmed that TEM8 is specifically expressed in tumor vascular endothelial cells.However,so far,the expression characteristics and potential significance of TEM8 in endothelial cells of clinically common solid tumors have not been clear.In addition,whether TEM8 is involved in tumor angiogenesis and its mechanism in tumor angiogenesis remains to be clarified.In this study,human lung cancer tissues were selected as the study object,and the expression of TEM8 in vascular endothelium of 58 cases of non-small cell lung cancer and adjacent tissues was detected by immunohistochemical staining.Furthermore,the relationship between the microvascular count with positive TEM8 expression in the interstitium of lung cancer and the clinicopathological characteristics was analyzed.The interstitial blood vessels of lung cancer were stained with TEM8 and CD34 by immunofluorescence double staining.To further clarify the specific expression of TEM8 in tumor endothelial cells and its possible mechanism of action in tumor angiogenesis,tumor endothelial cells 2H11 were selected for in vitro cytological experiments.HUVEC of human umbilical vein endothelial cells was used as the control,and the expression of TEM8 in tumor endothelial cells was detected by cellular immunofluorescence,flow cytometry,Western Blot and RT-PCR.Furthermore,2H11 cells were treated with IL-1?,and molecules related to the WNT signaling pathway were detected by Western Blot and RT-PCR aiming to preliminarily explore the molecular mechanism of TEM8 promoting the proliferation of tumor endothelial cells by activating the WNT signaling pathway.The proliferation ability of 2H11 cells before and after IL-1? stimulation was detected by CCK-8 assay,and the cell migration ability was observed by scratch assay.The results are as follows:? ? the expression of TEM8 in NSCLC tissues and its relationship with clinicopathological features1.Immunohistochemical results showed that TEM8 was mainly located in the cytoplasm and/or cell membrane of the interstitial vascular endothelial cells of lung cancer,and could be distributed completely or scattered along the lumen of the microvessel.The positive rate of TEM8 expression in the tumor tissues of NSCLC patients was 96.56%(56/58),and the positive rate in the adjacent tissues was 20.69%(12/58)showing a significant difference between the two(P<0.05),indicating that TEM8 was specifically expressed in the interstitial vascular endothelium of lung cancer.2.The TEM8-positive expression area in the lung cancer stroma was counted and the relationship between the TEM8-positive microvascular density and the clinicopathological characteristics of NSCLC patients was analyzed.The results showed that the microvascular density of TEM8 in patients with advanced clinical stage was significantly higher than that in patients with early stage(P=0.0011).The density of TEM8 microvessels in patients with lymph node metastasis was significantly higher than that in patients without lymph node metastasis(P<0.0001).There was no significant correlation with age,gender,tumor size and tumor differentiation(P>0.05).3.According to the TEM8 staining criteria,the proportion of positive cells and staining intensity of TEM8 in the stroma of lung cancer were statistically analyzed semi-quantitatively.CD34 was used to mark tumor blood vessels,MVD value was counted,and the correlation between TEM8 and CD34 expression was analyzed.The results showed that the MVD value of CD34 positive in tumor tissues of NSCLC patients with high expression of TEM8 was significantly higher than that of those with low expression of TEM8(P<0.05).4.Immunofluorescence double staining was used to localize the expression of TEM8 and CD34 in the interstitial vessels of lung cancer.The results showed that TEM8 and CD34 labeled vessels were co-located in the stroma of lung cancer,and could be expressed in different vascular endothelium.??Specific expression of TEM8 in tumor endothelial cells and its possible mechanism of tumor angiogenesis1.Tumor endothelial cells 2H11 were used for in vitro cytological experiments,and human umbilical vein endothelial cells HUVEC were used as control.The expression of TEM8 in tumor endothelial cells was detected from protein and m RNA levels.Results of cellular immunofluorescence,flow cytometry,Western Blot and RTPCR all showed that TEM8 was specifically expressed in tumor endothelial cells,while it was negative or weakly expressed in normal endothelial cells.2.IL-1? was used to treat 2H11 cells,and Western Blot,RT-PCR and other methods were used to detect the expression of related molecules in the WNT signaling pathway,including the expression of ?-catenin,phosphorylated ?-catenin,Axin-2 and Cyclin D1.The results showed that IL-1? stimulated 2H11 cells at 10ng/m L,and the expression of TEM8 was the highest at 2h.After IL-1? treatment,the expression of TEM8 at both protein and RNA levels was up-regulated.In addition,the expression of related molecules in wnt signaling pathway,including ?-catenin,Axin-2 and Cyclin D1,was significantly increased at the protein and RNA levels.The level of phosphorylated ?-catenin protein was significantly reduced(P<0.05),indicating increased activity of the wnt signaling pathway.3.The results of CCK-8 cell proliferation assay and scratch assay showed that after 10ng/m L IL-1? treatment of 2H11 cells for 6h and 12 h,up-regulated TEM8 expression could promote TECs proliferation.After treatment with 10ng/m L IL-1? for 24 h,the cell migration rate increased significantly(P<0.01),indicating that up-regulation of TEM8 expression could promote TECs migration.Through the above experiments,the following conclusions are drawn: TEM8 is specifically expressed in the intermal vascular endothelium of lung cancer,and its expression is closely related to lymph node metastasis and clinicopathological stage of lung cancer.Lung cancer tissues with high TEM8 expression had significantly higher CD34 positive microvascular density and low TEM8 expression.TEM8 may promote proliferation,migration,and angiogenesis of tumor endothelial cells by activating the wnt-?-catenin signaling pathway.The clarification of the above questions provides experimental basis for the use of TEM8 as a tumor vascular specific marker and for the specific tumor treatment targeting TEM8.It also provides a new idea to study tumor angiogenesis mechanism with TEM8 as the entry point. |
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